WO2020146625A1 - Polypeptides du complexe 2 de la sclérose tubéreuse modifiés - Google Patents

Polypeptides du complexe 2 de la sclérose tubéreuse modifiés Download PDF

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WO2020146625A1
WO2020146625A1 PCT/US2020/012924 US2020012924W WO2020146625A1 WO 2020146625 A1 WO2020146625 A1 WO 2020146625A1 US 2020012924 W US2020012924 W US 2020012924W WO 2020146625 A1 WO2020146625 A1 WO 2020146625A1
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cell
tsc2
seq
polypeptide
engineered
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PCT/US2020/012924
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David A. Kass
Mark J. RANEK
Kristen KOKKONEN
Brittany DUNKERLY-EYRING
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The Johns Hopkins University
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Priority to US17/421,249 priority Critical patent/US20220118017A1/en
Priority to EP20738372.0A priority patent/EP3908662A4/fr
Publication of WO2020146625A1 publication Critical patent/WO2020146625A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0657—Cardiomyocytes; Heart cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A61K35/17—Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A61P11/06—Antiasthmatics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P33/00—Antiparasitic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2510/00—Genetically modified cells

Definitions

  • This document relates to engineered tuberous sclerosis complex 2 (TSC2) polypeptides in which the ability of a residue (e.g., a residue corresponding to a serine residue in a wild type TSC2 polypeptide) to be phosphorylated is altered.
  • a residue e.g., a residue corresponding to a serine residue in a wild type TSC2 polypeptide
  • an engineered TSC2 polypeptide cannot be phosphorylated (e.g., by substituting a serine residue with an alanine residue).
  • an engineered TSC2 polypeptide can act as if it is constitutively phosphorylated (e.g., by substituting a serine residue with a glutamic acid residue).
  • rapamycin complex 1 The mechanistic target of rapamycin complex 1 (mTORCl) coordinates biosynthetic and recycling pathways to control cell growth and metabolic homeostasis (Dibble et al, 2013 Nat Cell Biol 15:555; and Wolfson et al, 2016 Science 351 :43).
  • mTORCl stimulates anabolic growth and suppresses protein recycling by autophagy.
  • mTORCl contributes to disease such as autoimmune disorders, cancer, and heart failure (Sciarretta et al. , 2014 Circ Res 114:549), where its hyperactivation is a therapeutic target (Laplante et al, 2012 Cell 149:274).
  • TSC2 Tuberous Sclerosis Complex 2
  • tuberin a GTPase activating protein that modifies Rheb-GTP binding to stimulate or suppress mTORCl
  • TSC2 is constitutively inhibitory, as gene deletion and loss-of-function mutations induce mTORCl hyperactivity, causing tumors and neurological disease.
  • ERK1/2 extra-cellular response kinase
  • Akt protein kinase B
  • p90Rsk reduce TSC2 inhibition
  • AMPK energy depletion-stimulated AMP-activated protein kinase
  • GSK-3 glycogen synthase kinase-3
  • TCR T-Cell Receptor
  • the mTORCl signaling pathway is engaged and ultimately determines the outcome of antigen recognition and cellular signaling response to the immune microenvironment.
  • T-cells stimulation of mTORCl results in enhancement of their effector function. This can involve clonal expansion so that the population size of a specific antigen-receptive cell is amplified to combat a foreign (or perceived to be foreign as in the case of autoimmune disease) body. It can also involve the enhanced synthesis and release of cytokines that coordinate an immune response. Stimulation of mTORCl also enhances cytotoxic responses controlled by T cells to target foreign cells (e.g . tumor cells, virus, bacteria, other foreign bodies) with the goal of eliminating these cells from the host.
  • foreign cells e.g . tumor cells, virus, bacteria, other foreign bodies
  • T-cells the suppression of mTORCl activity allows cells to remain in a more undifferentiated state, where they can replicate while still maintaining full differentiation potential. Reducing activity also enhances memory/persistence in effector T-cells and reduces the development of immunological anergy and exhaustion.
  • immunological self-recognition e.g. control over the immune system to recognize self from foreign cells and cell products. This is principally controlled by regulatory T-cells (Treg) that are central for suppressing immune reactions to self-antigens.
  • Treg regulatory T-cells
  • mTORCl stimulation and inhibition also play roles in the modulation of inflammatory cells, such as neutrophils and macrophages. These cells are commonly engaged in autoimmune disease where identification of a self-antigen has resulted in the activation of an immunological response, and local release of cytokines and other factors stimulates inflammatory cells to attack the body and cause disease.
  • inflammatory cells such as neutrophils and macrophages.
  • neutrophils and macrophages These cells are commonly engaged in autoimmune disease where identification of a self-antigen has resulted in the activation of an immunological response, and local release of cytokines and other factors stimulates inflammatory cells to attack the body and cause disease.
  • the regulation of neutrophil and macrophage function to the corresponding inflammatory response also depends on their ability to control cell growth, metabolism, and protein homeostasis, and mTORCl plays a central role to these factors and thus the functionality of these inflammatory cells.
  • TSC2 polypeptides in which the ability of a residue (e.g., a residue corresponding to a serine residue in a wild type TSC2 polypeptide) to be phosphorylated (e.g., as compared to a wild-type TSC2 polypeptide) is altered.
  • a TSC2 polypeptide can be engineered at a serine residue corresponding to SI 365 of the human TSC2 polypeptide sequence (SEQ ID NO: 5).
  • a TSC2 polypeptide can be engineered at a serine residue corresponding to SI 365, and, optionally, S1364 of the human TSC2 polypeptide sequence (SEQ ID NO: 5).
  • an engineered TSC2 polypeptide can include a substitution at residue S1365 (e.g., a substitution of the serine residue with an alanine residue (e.g., S1365A)) that cannot be phosphorylated, and, optionally, a substitution at residue S1364 (e.g., a substitution of the serine residue with an alanine residue (e.g., S1364A)) that cannot be phosphorylated.
  • an engineered TSC2 polypeptide includes a substitution at residue S1365 (e.g., S1365A) that cannot be phosphorylated, and, optionally, a substitution at residue S1364 (e.g., S13654) that cannot be phosphorylated.
  • an engineered TSC2 polypeptide can include a substitution at residue SI 365 (e.g., a substitution of the serine residue with a glutamic acid residue (e.g., S1365E)) that can act as if it is constitutively phosphorylated, and, optionally, a substitution at residue SI 364 (e.g., a substitution of the serine residue with a glutamic acid residue (e.g., S1364E)) that can act as if it is constitutively phosphorylated.
  • SI 365 e.g., a substitution of the serine residue with a glutamic acid residue (e.g., S1365E)
  • SI 364 e.g., a substitution of the serine residue with a glutamic acid residue (e.g., S1364E)
  • an engineered TSC2 polypeptide includes a substitution at residue SI 365 (e.g., S1365E) that can act as if it is constitutively phosphorylated, and, optionally, a substitution at residue SI 364 (e.g., S1365E) that can act as if it is constitutively phosphorylated.
  • SI 365 e.g., S1365E
  • SI 364 e.g., S1365E
  • This document also provides methods for making and using the engineered TSC2 polypeptides described herein, as well as engineered immune cells including engineered TSC2 polypeptides described herein and/or nucleic acid sequences encoding engineered TSC2 polypeptides described herein and methods of making and using such engineered immune cells.
  • phosphorylation of the serine residue S1365 in the human TSC2 polypeptide set forth in SEQ ID NO: 5, and, optionally, phosphorylation of the serine residue S1364 in the human TSC2 polypeptide set forth in SEQ ID NO: 5, confer potent effects on mTORCl stimulation. Neither modification alters basal mTORCl function. Having the ability to engineer TSC2 polypeptides provides a unique and unrealized opportunity to modify (e.g., increase or decrease) mTORCl signaling and to treat humans a disease or disorder characterized by aberrant mTORCl signaling.
  • one aspect of this document features a polypeptide comprising SEQ ID NO: 1.
  • this document features a nucleic acid encoding a polypeptide comprising SEQ ID NO: 1.
  • the nucleic acid can include the sequence of SEQ ID NO: 3.
  • this document features a vector including a nucleic acid encoding a polypeptide comprising SEQ ID NO: 1 (e.g., SEQ ID NO: 3).
  • this document features a cell comprising a vector including a nucleic acid encoding a polypeptide comprising SEQ ID NO: 1 (e.g., SEQ ID NO: 3).
  • the nucleic acid encoding the polypeptide comprising SEQ ID NO: 1 can be operably linked to a nucleic acid that drives expression of the polypeptide in the cell.
  • the cell can be an immune cell.
  • the immune cell can be a cytotoxic T cell or a chimeric antigen receptor T cell (CAR-T cell). Upon activation, the cytotoxic T cell or CAR-T cell can exhibit a higher level of mTORCl signaling than a reference cytotoxic T cell or a reference CAR-T cell that lacks the vector.
  • the cytotoxic T cell or CAR- T cell can express one or more cytokines at a higher level than a reference cytotoxic T cell or a reference CAR-T cell that lacks the vector, where the one or more cytokines can be interferon gamma, tumor necrosis factor alpha, interleukin 2, or combinations thereof.
  • the immune cell can be a helper T cell.
  • the helper T cell can exhibit a higher level of mTORCl signaling than a reference helper T cell that lacks the vector.
  • the immune cell can be a regulatory T cell.
  • the regulatory T cell can exhibit a higher level of mTORCl signaling than a reference regulatory T cell that lacks the vector.
  • the cell also can include a genetic alteration in which a wild type nucleic acid sequence encoding TSC2 has been rendered inactive.
  • this document features cell comprising a vector that includes a nucleic acid encoding a mutant TSC2 polypeptide, where the mutant TSC2 polypeptide includes an altered amino acid at a position corresponding to SI 365 of SEQ ID NO: 5, S1366 of SEQ ID NO: 6, or S1367 of SEQ ID NO: 7.
  • the altered amino acid can be a methionine, alanine, valine, leucine, isoleucine, or phenylalanine.
  • the mutant TSC2 polypeptide can include an amino acid sequence as set forth in one of SEQ ID NOs: 1, 8, or 9.
  • the nucleic acid encoding the mutant TSC2 polypeptide can be operably linked to a nucleic acid that drives expression of the mutant TSC2 polypeptide in the cell.
  • this document features a polypeptide comprising SEQ ID NO:
  • this document features a nucleic acid encoding a polypeptide comprising SEQ ID NO: 2.
  • the nucleic acid can include the sequence of SEQ ID NO: 4.
  • this document features a vector including a nucleic acid encoding a polypeptide comprising SEQ ID NO: 2 (e.g., SEQ ID NO: 4).
  • this document features a cell comprising a vector including a nucleic acid encoding a polypeptide comprising SEQ ID NO: 2 (e.g., SEQ ID NO: 4).
  • the nucleic acid encoding the polypeptide comprising SEQ ID NO: 2 can be operably linked to a nucleic acid that drives expression of the polypeptide in the cell.
  • the cell can be an immune cell.
  • the immune cell can be a memory T cell.
  • the memory T cell can exhibit a lower level of mTORCl signaling than a reference memory T cell that lacks the vector.
  • the memory T cell can express one or more cytokines at a lower level than a reference memory T cell that lacks the vector, where the one or more cytokines can be interferon gamma, tumor necrosis factor alpha, interleukin 2, or combinations thereof.
  • the cell also can include a genetic alteration in which a wild type nucleic acid sequence encoding TSC2 has been rendered inactive.
  • this document features a cell comprising a vector that includes a nucleic acid encoding a mutant TSC2 polypeptide.
  • the mutant TSC2 polypeptide can include an altered amino acid at a position corresponding to S1365 of SEQ ID NO: 5, S1366 of SEQ ID NO: 6, or S1367 of SEQ ID NO: 7.
  • the altered amino acid can be aspartic acid or glutamic acid.
  • the mutant TSC2 polypeptide can include an amino acid sequence as set forth in one of SEQ ID NOs: 2, 10, or 11.
  • the nucleic acid encoding the mutant TSC2 polypeptide can be operably linked to a nucleic acid that drives expression of the mutant TSC2 polypeptide in the cell.
  • this document features a method of treating a disease in a subject in need thereof.
  • the methods can include, or consist essentially of, administering to a subject an engineered immune cell comprising a vector that includes a nucleic acid encoding a polypeptide including SEQ ID NO: 1 operably linked to a nucleic acid that drives expression of the polypeptide in the T cell, and where, upon recognizing an antigen associated with the disease, the immune cell can exhibit increased activity as compared to a reference immune cell that lacks the vector.
  • the engineered immune cell can be a cytotoxic T cell.
  • the increased activity of the cytotoxic T cell can include increased mTORCl signaling.
  • the increased activity of the cytotoxic T cell can include increased expression of one or more cytokines selected from the group consisting of: interferon gamma, tumor necrosis factor alpha, interleukin 2, and combinations thereof.
  • the engineered immune cell can be a helper T cell.
  • the increased activity of the helper T cell can include increased mTORCl signaling.
  • the disease can be cancer, a viral disease, a bacterial disease, fungal disease, or a parasitic disease.
  • the engineered immune cell can be a regulatory T cell.
  • the increased activity of the regulatory T cell can include increased mTORCl signaling.
  • the disease can be asthma, an autoimmune disease, or graft vs. host disease.
  • the engineered immune cell can include a genetic alteration in a wild type nucleic acid sequence encoding TSC2, where the genetic alteration renders the TSC2 inactive.
  • the engineered immune cell can be derived from an endogenous immune cell obtained from the subject.
  • this document features a method of generating a persistent T cell in a subject.
  • the methods can include, or consist essentially of, administering to a subject an engineered immune cell comprising a vector that includes a nucleic acid encoding a polypeptide comprising SEQ ID NO: 2 operably linked to a nucleic acid that drives expression of the polypeptide in the engineered immune cell, where the engineered immune cell recognizes an antigen, where, upon recognizing the antigen, the engineered immune cell can exhibit decreased activity as compared to a reference immune cell that lacks the vector, and where, upon recognizing the antigen, the engineered immune cell can be become the persistent T cell.
  • the decreased activity of the engineered immune cell can include decreased mTORCl signaling.
  • the engineered immune cell can be derived from an endogenous immune cell obtained from the subject.
  • the engineered immune cell can be a CD8+ T cell, and the persistent T cell can be a memory T cell.
  • the CD8+ T cell can be further engineered to express a chimeric antigen receptor or a T cell receptor.
  • the engineered immune cell can be a regulatory T cell, and the persistent T cell can be a persistent T regulatory cell.
  • this document features a method of generating a persistent T cell in vitro.
  • the methods can include, or consist essentially of, providing an immune cell, introducing into the immune cell a vector including a nucleic acid encoding a polypeptide comprising SEQ ID NO: 2 operably linked to a nucleic acid that drives expression of the polypeptide in the immune cell, thereby generating an engineered immune cell, where the engineered immune cell exhibits decreased mTORCl signaling as compared to a reference immune cell that lacks the vector, contacting the engineered immune cell with an antigen that is recognized by the engineered immune cell, and culturing the engineered immune cell under conditions and for a time sufficient such that the engineered immune cell becomes the persistent T cell.
  • the immune cell can be a CD8+ T cell, and the persistent T cell can be a memory T cell.
  • the CD8+ T cell can be further engineered to express a chimeric antigen receptor or a T cell receptor.
  • the method also can include administering the memory T cell to a subject.
  • the subject can exhibit a disease, and the administration of the memory T cell to the subject can treat the disease.
  • the disease can be cancer, a viral disease, a bacterial disease, fungal disease, or a parasitic disease.
  • the immune cell can be obtained from the subject.
  • the immune cell can be a regulatory T cell, and the persistent T cell can be a persistent T regulatory cell.
  • the method also can include administering the persistent T regulatory cell to a subject.
  • the subject can exhibit a disease, and the administration of the persistent T regulatory cell to the subject can treat the disease.
  • the disease can be asthma, an autoimmune disease, or graft vs. host disease.
  • the immune cell can be obtained from the subject.
  • this document features a polypeptide including SEQ ID NO: 20.
  • this document a nucleic acid encoding a polypeptide including SEQ ID NO: 20.
  • the nucleic acid can include the sequence of SEQ ID NO: 22.
  • this document features a vector including a nucleic acid encoding a polypeptide including SEQ ID NO: 20 (e.g., SEQ ID NO: 22).
  • this document features a polypeptide including SEQ ID NO: 21.
  • this document a nucleic acid encoding a polypeptide including SEQ ID NO: 21.
  • the nucleic acid can include the sequence of SEQ ID NO: 23.
  • this document features a vector including a nucleic acid encoding a polypeptide including SEQ ID NO: 21 (e.g., SEQ ID NO: 23).
  • FIG. 1 shows that protein kinase G (PKG) activates autophagic flux in isolated cardiac myocytes.
  • ET1 stimulates myocyte growth (hypertrophy) and this is markedly reduced by protein kinase G stimulation.
  • These cells display an increase in red dots indicating enhanced autophagy.
  • Figure 2 contains a phospho-site map for TSC2, with numbering based on the human polypeptide sequence. Sequences shown include residues 1357-1395 of the human TSC2 sequence set forth in SEQ ID NO: 5, and residues 1358 -1396 of the mouse TSC2 sequence set forth in SEQ ID NO: 6. These sites are all highly homologous between human and other mammalian species, though the numbering is slightly different for the T1271 AMP-kinase (AMPK) site (for mouse it is T1270), and for all sites identified at and above S1364 (mouse numbering is one higher for each; e.g., human S1364 (hsS1364) is equivalent to mouse S1365 (mmS1365)).
  • AMPK AMP-kinase
  • FIGS 3A and 3B show that myocytes stimulated with a growth hormone - endothelin-1 exhibit increased protein kinase G activation, and that this results in the phosphorylation of TSC2 at either hsS1364 or hsS1365. The latter is equally blunted by site mutagenesis of the serine to either an alanine (SA) or glutamic acid (SE) at either hsS1364 or hsS1365.
  • SA alanine
  • SE glutamic acid
  • Figure 4 shows Nppb mRNA expression (pathological hypertrophy gene marker) in myocytes transfected with WT, SA, or SE TSC2 (huS1365 mutated), and then exposed to 48-hrs ET1 to induce hypertrophy.
  • Activation of PKG by sildenafil (Sil) reduces Nppb increase in WT expressing cells, but not those with SA or SE.
  • SE expression depresses Nppb rise, whereas SA expression enhances it.
  • the results are shown for two sets of experiments. On the left, both the SA and SE mutations targeted hsS1364 (mmS1365). On the right, both the SA and SE mutations targeted hsS1365 (mmS1366).
  • Figure 5 shows mTORCl signaling proteins and for markers of autophagy (p62 and LC3-II) when hsS1364 (mmS1365) was mutated to either alanine (SA) or glutamate (SE) (upper set of data), and when hsS1365 (mmS1366) was mutated to either alanine (SA) or glutamate (SE) (lower set of data).
  • Western blots are from the same experiment as shown in Figure 4.
  • Expression of hsS1364E or hsS1365E reduces mTORCl activation and p62, while further increasing LC3-II.
  • expression of hsS1364A or hsS1365A further increased mTORCl activation (more p70S6K and 4EBP1
  • Figure 7 contains an amino acid sequence (SEQ ID NO: 1) of an exemplary engineered TSC2 polypeptide having an alanine substitution at the serine residue at position S1365 of the human TSC2 polypeptide sequence.
  • Figure 8 contains an amino acid sequence (SEQ ID NO: 2) of an exemplary engineered TSC2 polypeptide having a glutamic acid substitution at the serine residue at position S1365 of the human TSC2 polypeptide sequence.
  • Figure 9 contains a nucleic acid sequence (SEQ ID NO: 3) encoding an engineered TSC2 polypeptide having an alanine substitution at the serine residue at position S1365 of the human TSC2 polypeptide sequence.
  • Figure 10 contains a nucleic acid sequence (SEQ ID NO: 4) encoding an engineered TSC2 polypeptide having a glutamic acid substitution at the serine residue at position S1365 of the human TSC2 polypeptide sequence.
  • Figure 11 contains an amino acid sequence (SEQ ID NO: 5) of an exemplary wild-type human TSC2 polypeptide sequence.
  • Figure 12 contains an amino acid sequence (SEQ ID NO: 6) of an exemplary wild-type mouse TSC2 polypeptide sequence.
  • Figure 13 contains an amino acid sequence (SEQ ID NO: 7) of an exemplary wild-type rat TSC2 polypeptide sequence.
  • Figure 14 contains an amino acid sequence (SEQ ID NO: 8) of an exemplary engineered TSC2 polypeptide having an alanine substitution at the serine residue at position S1366 of the mouse TSC2 polypeptide sequence.
  • Figure 15 contains an amino acid sequence (SEQ ID NO: 9) of an exemplary engineered TSC2 polypeptide having an alanine substitution at the serine residue at position S1367 of the rat TSC2 polypeptide sequence.
  • Figure 16 contains an amino acid sequence (SEQ ID NO: 10) of an exemplary engineered TSC2 polypeptide having a glutamic acid substitution at the serine residue at position S1366 of the mouse TSC2 polypeptide sequence.
  • Figure 17 contains an amino acid sequence (SEQ ID NO: 11) of an exemplary engineered TSC2 polypeptide having a glutamic acid substitution at the serine residue at position S1367 of the rat TSC2 polypeptide sequence.
  • Figure 18 contains an amino acid sequence (SEQ ID NO: 12) of an exemplary engineered TSC2 polypeptide having an alanine substitution at the serine residue at position S1364 of the human TSC2 polypeptide sequence.
  • Figure 19 contains an amino acid sequence (SEQ ID NO: 13) of an exemplary engineered TSC2 polypeptide having a glutamic acid substitution at the serine residue at position S1364 of the human TSC2 polypeptide sequence.
  • Figure 20 contains a nucleic acid sequence (SEQ ID NO: 14) encoding an amino acid sequence of an exemplary engineered TSC2 polypeptide having an alanine substitution at the serine residue at position SI 364 of the human TSC2 polypeptide sequence.
  • Figure 21 contains a nucleic acid sequence (SEQ ID NO: 15) encoding an amino acid sequence of an exemplary engineered TSC2 polypeptide having a glutamic acid substitution at the serine residue at position SI 364 of the human TSC2 polypeptide sequence.
  • Figure 22 contains an amino acid sequence (SEQ ID NO: 16) of an exemplary engineered TSC2 polypeptide having an alanine substitution at the serine residue at position S1365 of the mouse TSC2 polypeptide sequence.
  • Figure 23 contains an amino acid sequence (SEQ ID NO: 17) of an exemplary engineered TSC2 polypeptide having an alanine substitution at the serine residue at position S1366 of the rat TSC2 polypeptide sequence.
  • Figure 24 contains an amino acid sequence (SEQ ID NO: 18) of an exemplary engineered TSC2 polypeptide having a glutamic acid substitution at the serine residue at position S1365 of the mouse TSC2 polypeptide sequence.
  • Figure 25 contains an amino acid sequence (SEQ ID NO: 19) of an exemplary engineered TSC2 polypeptide having a glutamic acid substitution at the serine residue at position S1366 of the rat TSC2 polypeptide sequence.
  • Figure 26 contains an amino acid sequence (SEQ ID NO: 20) of an exemplary engineered TSC2 polypeptide having an alanine substitution at the serine residue at position S1364 and having an alanine substitution at the serine residue at position S1365 of the human TSC2 polypeptide sequence.
  • Figure 27 contains an amino acid sequence (SEQ ID NO: 21) of an exemplary engineered TSC2 polypeptide having a glutamic acid substitution at the serine residue at position S1364 and having a glutamic acid substitution at the serine residue at position SI 365 of the human TSC2 polypeptide sequence.
  • Figure 28 contains a nucleic acid sequence (SEQ ID NO: 22) encoding an amino acid sequence of an exemplary engineered TSC2 polypeptide having an alanine substitution at the serine residue at position SI 364 and having an alanine substitution at the serine residue at position S1365 of the human TSC2 polypeptide sequence.
  • Figure 29 contains a nucleic acid sequence (SEQ ID NO: 23) encoding an amino acid sequence of an exemplary engineered TSC2 polypeptide having a glutamic acid substitution at the serine residue at position SI 364 and having a glutamic acid substitution at the serine residue at position SI 365 of the human TSC2 polypeptide sequence.
  • Figure 30 contains an amino acid sequence (SEQ ID NO: 24) of an exemplary engineered TSC2 polypeptide having an alanine substitution at the serine residue at position S1365 and having an alanine substitution at the serine residue at position S1366 of the mouse TSC2 polypeptide.
  • Figure 31 contains an amino acid sequence (SEQ ID NO: 25) of an exemplary engineered TSC2 polypeptide having an glutamic acid substitution at the serine residue at position S1365 and having an glutamic acid substitution at the serine residue at position SI 366 of the mouse TSC2 polypeptide.
  • Figure 32 contains an amino acid sequence (SEQ ID NO: 26) of an exemplary engineered TSC2 polypeptide having an alanine substitution at the serine residue at position S1366 and having an alanine substitution at the serine residue at position S1367 of the rat TSC2 polypeptide.
  • Figure 33 contains an amino acid sequence (SEQ ID NO: 27) of an exemplary engineered TSC2 polypeptide having an glutamic acid substitution at the serine residue at position S1366 and having an glutamic acid substitution at the serine residue at position SI 367 of the rat TSC2 polypeptide.
  • TSC2 polypeptides in which the ability of a residue (e.g., a residue corresponding to a serine residue in a wild type TSC2 polypeptide) to be phosphorylated (e.g., as compared to a wild-type TSC2 polypeptide) is altered.
  • a TSC2 polypeptide can be engineered at a serine residue corresponding to SI 365 of the human TSC2 polypeptide sequence (SEQ ID NO: 5), and, optionally, can also be engineered at a serine residue corresponding to S1364 of the human TSC2 polypeptide sequence (SEQ ID NO: 5).
  • an engineered TSC2 polypeptide can exhibit a decreased ability to be phosphorylated.
  • an engineered TSC2 polypeptide having a substitution at residue SI 365 e.g., a substitution of the serine residue with an alanine residue (S1365A)
  • a substitution at residue S13645 e.g., a substitution of the serine residue with an alanine residue (S1364A)
  • can exhibit a decreased ability to be phosphorylated e.g, cannot be phosphorylated.
  • an engineered TSC2 polypeptide having a substitution at residue S1365 e.g., S1365A
  • S1365A e.g., S1365A
  • S1364 e.g., S 1365 A
  • an engineered TSC2 polypeptide having a substitution at residue S1365 can exhibit a decreased ability to be phosphorylated (e.g., cannot be phosphorylated).
  • an engineered TSC2 polypeptide can be pseudo-phosphorylated.
  • an engineered TSC2 polypeptide having a substitution at residue SI 365 e.g, a substitution of the serine residue with a glutamic acid residue (S1365E)
  • a substitution at residue S1364 e.g., a substitution of the serine residue with a glutamic acid residue (S1364E)
  • can be pseudo-phosphorylated e.g, can act as if it is constitutively phosphorylated.
  • an engineered TSC2 polypeptide having a substitution at residue S1365 e.g., S1365E
  • S1364 e.g., S1364E
  • S1365E residue S1365E
  • S1364E residue S1364E
  • This document also provides methods for making and using the engineered TSC2 polypeptides described herein, as well as engineered immune cells including engineered TSC2 polypeptides described herein and/or nucleic acid sequences encoding engineered TSC2 polypeptides described herein and methods of making and using such engineered immune cells.
  • the word“a” before a noun represents one or more of the particular noun.
  • the phrase“a genetic alteration” encompasses“one or more genetic alterations.”
  • the term“about” means approximately, in the region of, roughly, or around. When used in conjunction with a numerical range, the term“about” modifies that range by extending the boundaries above and below the numerical values set forth.
  • the term“subject” means a vertebrate, including any member of the class mammalia, including humans, domestic and farm animals, and zoo, sports or pet animals, such as mouse, rabbit, pig, sheep, goat, cattle, horse (e.g., race horse), and higher primates.
  • the subject is a human.
  • the subject has a disease.
  • the subject has cancer.
  • the subject has a viral disease. In some embodiments, the subject has a bacterial disease. In some embodiments, the subject has a fungal disease. In some embodiments, the subject has a parasitic disease. In some embodiments, the subject has asthma. In some embodiments, the subject has an autoimmune disease. In some embodiments, the subject has graft vs. host disease.
  • engineered TSC2 polypeptides that cannot be phosphorylated at a residue that has been substituted for a serine residue, or that cannot be phosphorylated to the extent that a non-engineered TSC2 polypeptide having the serine residue can be phosphorylated.
  • engineered TSC2 polypeptides having an amino acid substitution at a residue corresponding to the serine residue at position S1365 of the human TSC2 polypeptide sequence (SEQ ID NO: 5).
  • the serine residue at position S1365 of SEQ ID NO: 5 (or a corresponding amino acid residue in a different TSC2 polypeptide) is substituted with an amino acid with an aliphatic side chain.
  • engineered TSC2 polypeptides provided herein have an amino acid substitution at a residue at (or at an amino acid corresponding to) the serine residue at position SI 364 and/or SI 365 of the human TSC2 polypeptide sequence (SEQ ID NO: 5).
  • the serine residue at position S1364 and/or SI 365 of SEQ ID NO: 5, or a serine residue in a TSC2 polypeptide that corresponds to these serine residues is/are substituted with an amino acid with an aliphatic side chain.
  • the serine residue at position S1365 of SEQ ID NO: 5 is substituted with a methionine residue, an alanine residue, a valine residue, a leucine residue, an isoleucine residue, or a phenylalanine residue.
  • the serine residue at position S1364 and/or S1365 of SEQ ID NO: 5, or a serine residue in a polypeptide that corresponds to these serine residues is/are substituted with a methionine residue, an alanine residue, a valine residue, a leucine residue, an isoleucine residue, or a phenylalanine residue.
  • an engineered TSC2 polypeptide having an alanine substitution at the serine residue at position SI 365 of the human TSC2 polypeptide sequence is provided (e.g., SEQ ID NO: 1).
  • an engineered TSC2 polypeptide having alanine substitutions at the serine residue positions S1364 and S1365 of the human TSC2 polypeptide sequence is provided (e.g., SEQ ID NO: 20).
  • engineered TSC2 polypeptides disclosed herein cannot be phosphorylated to the extent that a non-engineered TSC2 polypeptide having the serine residue(s) can be phosphorylated.
  • engineered TSC2 polypeptides disclosed herein are phosphorylated to an extent that is about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or less than a non-engineered TSC2 polypeptide having the serine residue(s). In some embodiments, engineered TSC2 polypeptides disclosed herein cannot be phosphorylated.
  • engineered TSC2 polypeptides having an amino acid substitution at a residue corresponding to the serine residue at position S1366 of the mouse TSC2 polypeptide sequence (SEQ ID NO: 6) such that engineered TSC2 polypeptides cannot be phosphorylated at that residue, or cannot be phosphorylated to the extent that a non-engineered TSC2 polypeptide having the serine residue can be phosphorylated.
  • engineered TSC2 polypeptides provided herein have an amino acid substitution at a residue corresponding to the serine residue at position S1365 and/or S1366 of the mouse TSC2 polypeptide sequence (SEQ ID NO: 6) such that engineered TSC2 polypeptides cannot be phosphorylated at that residue, or cannot be phosphorylated to the extent that a non-engineered TSC2 polypeptide having the serine residue can be phosphorylated.
  • the serine residue at position S1366 of SEQ ID NO: 6 is substituted with an amino acid with an aliphatic side chain.
  • the serine residue at position S1365 and/or S1366 of SEQ ID NO: 6, or a serine residue in a TSC2 polypeptide that corresponds to these serine residue is/are substituted with an amino acid with an aliphatic side chain.
  • the serine residue at position S1366 of SEQ ID NO: 6 is substituted with a methionine residue, an alanine residue, a valine residue, a leucine residue, an isoleucine residue, or a phenylalanine residue.
  • the serine residue at position S1365 and/or S1366 of SEQ ID NO: 6, or a serine residue in a polypeptide that corresponds to these serine residues is substituted with a methionine residue, an alanine residue, a valine residue, a leucine residue, an isoleucine residue, or a phenylalanine residue.
  • an engineered TSC2 polypeptide having an alanine substitution at the serine residue at position SI 366 of the mouse TSC2 polypeptide sequence is provided (e.g., SEQ ID NO: 8).
  • an engineered TSC2 polypeptide having alanine substitutions at the serine residue positions SI 365 and SI 366 of the mouse TSC2 polypeptide sequence is provided (e.g., SEQ ID NO: 24).
  • engineered TSC2 polypeptides disclosed herein cannot be phosphorylated to the extent that a non-engineered TSC2 polypeptide having the serine residue(s) can be phosphorylated.
  • engineered TSC2 polypeptides disclosed herein are phosphorylated to an extent that is about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or less than a non- engineered TSC2 polypeptide having the serine residue(s). In some embodiments, engineered TSC2 polypeptides disclosed herein cannot be phosphorylated.
  • engineered TSC2 polypeptides having an amino acid substitution at a residue corresponding to the serine residue at position S1367 of the rat TSC2 polypeptide sequence (SEQ ID NO: 7) such that engineered TSC2 polypeptides cannot be phosphorylated at that residue, or cannot be phosphorylated to the extent that a non-engineered TSC2 polypeptide having the serine residue can be phosphorylated.
  • engineered TSC2 polypeptides have an amino acid substitution at a residue corresponding to the serine residue at position S1366 and/or S1367 of the rat TSC2 polypeptide sequence (SEQ ID NO: 7) such that engineered TSC2 polypeptides cannot be phosphorylated at that residue, or cannot be phosphorylated to the extent that a non-engineered TSC2 polypeptide having the serine residue can be phosphorylated.
  • the serine residue at position S1367 of SEQ ID NO: 7 is substituted with an amino acid with an aliphatic side chain.
  • the serine residue at position S1366 and/or S1377 of SEQ ID NO: 7, or a serine residue in a TSC2 polypeptide that corresponds to these serine residues is/are substituted with an amino acid with an aliphatic side chain.
  • the serine residue at position SI 367 of SEQ ID NO: 7 is substituted with a methionine residue, an alanine residue, a valine residue, a leucine residue, an isoleucine residue, or a phenylalanine residue.
  • the serine residue at position S1366 and/or S1367 of SEQ ID NO: 7, or a serine residue in a polypeptide that corresponds to these serine residues is/are substituted with a methionine residue, an alanine residue, a valine residue, a leucine residue, an isoleucine residue, or a phenylalanine residue.
  • an engineered TSC2 polypeptide having an alanine substitution at the serine residue at position S1367 of the rat TSC2 polypeptide sequence is provided (SEQ ID NO: 9).
  • an engineered TSC2 polypeptide having alanine substitutions at the serine residue positions S1366 and S1367 of the rat TSC2 polypeptide sequence is provided (SEQ ID NO: 26).
  • engineered TSC2 polypeptides disclosed herein cannot be phosphorylated to the extent that a non-engineered TSC2 polypeptide having the serine residue(s) can be phosphorylated.
  • engineered TSC2 polypeptides disclosed herein are phosphorylated to an extent that is about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or less than a non-engineered TSC2 polypeptide having the serine(s). In some embodiments, engineered TSC2 polypeptides disclosed herein cannot be phosphorylated.
  • vectors that include nucleic acid sequences that encode polypeptides that cannot be phosphorylated at an amino acid position corresponding to S1364 and/or S1365 of the human TSC2 polypeptide sequence (SEQ ID NO: 5), S1365 and/or S1366 of the mouse TSC2 polypeptide sequence (SEQ ID NO: 6), or S1366 and/or SI 367 of the rat TSC2 polypeptides sequence (SEQ ID NO: 7), or that cannot be phosphorylated to the extent that a TSC2 polypeptide having a serine residue at these positions can be phosphorylated, and cells having such vectors.
  • a vector that includes nucleic acid sequences that encode polypeptides that cannot be phosphorylated at position corresponding to S1364 and/or S1365 of the human TSC2 polypeptide sequence (SEQ ID NO: 5), S1365 and/or S1366 of the mouse TSC2 polypeptide sequence (SEQ ID NO: 6), or SI 366 and/or SI 367 of the rat TSC2 polypeptide sequence (SEQ ID NO: 7), or that cannot be phosphorylated to the extent that a TSC2 polypeptide having a serine residue at these positions can be phosphorylated can be any appropriate type of vector.
  • vectors include, without limitation, plasmids (e.g., expression plasmids) and vectors (e.g, viral vectors such as lentiviral vectors, retroviral vectors, adenovirus vectors, and adeno- associated virus vectors).
  • plasmids e.g., expression plasmids
  • vectors e.g, viral vectors such as lentiviral vectors, retroviral vectors, adenovirus vectors, and adeno- associated virus vectors.
  • a vector that includes nucleic acid sequences that encode polypeptides that cannot be phosphorylated at position corresponding to S1364 and/or S1365 of the human TSC2 polypeptide sequence (SEQ ID NO: 5), S1365 and/or SI 366 of the mouse TSC2 polypeptide sequence (SEQ ID NO: 6), or SI 366 and/or SI 367 of the rat TSC2 polypeptides sequence (SEQ ID NO: 7), or that cannot be phosphorylated to the extent that a TSC2 polypeptide having a serine residue at these positions can be phosphorylated can be a lentiviral vector.
  • a vector that includes nucleic acid sequences that encode polypeptides that cannot be phosphorylated at position corresponding to S1364 and/or S1365 of the human TSC2 polypeptide sequence (SEQ ID NO: 5), S1365 and/or S1366 of the mouse TSC2 polypeptide sequence (SEQ ID NO: 6), or SI 366 and/or SI 367 of the rat TSC2 polypeptides sequence (SEQ ID NO: 7), or that cannot be phosphorylated to the extent that a TSC2 polypeptide having a serine residue at these positions can be phosphorylated also can include one or more additional features (e.g., one or more additional features to modulate polypeptide expression).
  • a vector described herein also includes a promoter
  • the promoter can be operably linked to a nucleic acid sequence that encodes polypeptides that cannot be phosphorylated at position corresponding to S1364 and/or S1365 of the human TSC2 polypeptide sequence (SEQ ID NO: 5), S1365 and/or S1366 of the mouse TSC2 polypeptide sequence (SEQ ID NO: 6), or S1366 and/or S1367 of the rat TSC2 polypeptides sequence (SEQ ID NO: 7), or that cannot be phosphorylated to the extent that a TSC2 polypeptide having a serine residue at these positions can be phosphorylated (e.g., such that the promoter can drive expression of the nucleic acid sequence).
  • a promoter can be any appropriate promoter.
  • a promoter can be a constitutive promoter.
  • a promoter can be a viral promoter.
  • a promoter can be an inducible promoter.
  • a promoter can be a cell-specific and/or tissue-specific promoter. Examples of promoters that can be used to drive expression of nucleic acid sequences that encode polypeptides that cannot be
  • a promoter can be as described elsewhere (see, e.g., Morgan et al, 2016 Biomedicines. 4:9).
  • cells having vectors that include nucleic acid sequences that encode engineered TSC2 polypeptides that cannot be phosphorylated at a position that corresponds to a wild-type serine residue in any of the polypeptides described herein (or that cannot be phosphorylated to the extent that a TSC2 polypeptide having a serine residue at these positions can be phosphorylated) do not express endogenous, wild-type TSC2.
  • nucleic acid sequence encoding wild-type TSC2 can be modified by any of a variety of genetic manipulation techniques known in the art including, but not limited to, CRISPR-based methods, TALEN-based methods, and other genetic targeting or recombination methods (see, e.g., Roth et al, 2018 Nature 559:405-409).
  • TSC2 polypeptides disclosed herein can be engineered at amino acid positions S1364 and/or S1365 of the human TSC2 polypeptide sequence (SEQ ID NO: 5), S1365 and/or S1366 of the mouse TSC2 polypeptide sequence (SEQ ID NO: 6), or S1366 and/or SI 367 of the rat TSC2 polypeptide sequence (SEQ ID NO: 7) with any mutation or modification that results in TSC2 being unable to be phosphorylated (or that results in a decreased ability of TSC2 to be phosphorylated) at the respective positions in human (SI 364, SI 365), mouse (SI 365, SI 366), or rat (SI 366, SI 367).
  • TSC2 polypeptides from other species can be engineered with any mutation or modification (e.g., a residue corresponding to human S1364 and/or S1365, mouse S1365 and/or SI 366, and/or rat SI 366 and/or SI 367) that results in TSC2 being unable to be phosphorylated (or that results in a decreased ability of TSC2 to be phosphorylated).
  • any mutation or modification e.g., a residue corresponding to human S1364 and/or S1365, mouse S1365 and/or SI 366, and/or rat SI 366 and/or SI 367
  • nucleic acids encoding engineered TSC2 polypeptides that cannot be phosphorylated at positions corresponding to a serine residue in the wild- type TSC2 polypeptide sequence, or that cannot be phosphorylated to the extent that a non-engineered TSC2 polypeptide having the serine residue can be phosphorylated.
  • a nucleic acid sequence encoding an engineered TSC2 polypeptide having an amino acid substitution at the serine residue at position S1365 of the human TSC2 polypeptide sequence is provided (e.g., the nucleic acid sequence of SEQ ID NO:
  • nucleic acid sequence encoding an engineered TSC2 polypeptide having an amino acid substitution at the serine residue at position SI 364 and SI 365 of the human TSC2 polypeptide sequence is provided (e.g., the nucleic acid sequence of SEQ ID NO: 22).
  • nucleic acids provided herein encode engineered TSC2 polypeptides in which the serine residue at position S1365 of SEQ ID NO: 5, or a serine residue in a TSC2 polypeptide that corresponds to the serine residues at this position, is substituted with an amino acid with an aliphatic side chain.
  • nucleic acids provided herein encode engineered TSC2 polypeptides in which the serine residue at position S1364 and/or S1365 of SEQ ID NO: 5, or position S 1365 and/or S 1366 of SEQ ID NO : 6, or position S 13666 and/or S 1367 of SEQ ID NO : 7, or a serine residue in a TSC2 polypeptide that corresponds to the serine residues at these positions, are substituted with an amino acid with an aliphatic side chain.
  • nucleic acids provided herein encode engineered TSC2 polypeptides in which the serine residue at position S1364 and/or S1365 of SEQ ID NO: 5, or a serine residue in a TSC2 polypeptide that corresponds to the serine residues at this position, is substituted with a methionine residue, an alanine residue, a valine residue, a leucine residue, an isoleucine residue, or a phenylalanine residue.
  • nucleic acids provided herein encode engineered TSC2 polypeptides in which the serine residue at position S1364 and/or S1365 of SEQ ID NO: 5, or position S1365 and/or S1366 of SEQ ID NO: 6, or position S1366 and/or S1367 of SEQ ID NO: 7, or a serine residue in a TSC2 polypeptide that corresponds to the serine residues at these positions, are substituted with a methionine residue, an alanine residue, a valine residue, a leucine residue, an isoleucine residue, or a phenylalanine residue.
  • nucleic acids provided herein include the genetic codons of GCT, GCC, GCA, or GCG at positions that are translated to the amino acid alanine at position SI 365 of the human TSC2 polypeptide sequence (SEQ ID NO: 5).
  • nucleic acids provided herein include the genetic codons of GCT, GCC, GCA, or GCG at positions that are translated to the amino acid alanine at position S1364 and/or S1365 of the human TSC2 polypeptide sequence (SEQ ID NO: 5), or position S1365 and/or S1366 of the mouse TSC2 polypeptide sequence (SEQ ID NO: 6), or position S1366 and/or S1367 of the rat TSC2 polypeptide sequence (SEQ ID NO: 7).
  • nucleic acids provided herein include the genetic codons of GTT, GTC, GTA, or GTG at positions that are translated to the amino acid valine at position SI 365 of the human TSC2 polypeptide sequence (SEQ ID NO: 5), or position S1366 of the mouse TSC2 polypeptide sequence (SEQ ID NO: 6), or position S1367 of the rat TSC2 polypeptide sequence (SEQ ID NO: 7).
  • nucleic acids provided herein encode engineered TSC2 polypeptides that cannot be phosphorylated to the extent that a non-engineered TSC2 polypeptide having the serine residue(s) can be phosphorylated.
  • nucleic acids disclosed herein encode engineered TSC2 polypeptides that are
  • nucleic acids disclosed herein encode engineered TSC2 polypeptides that cannot be phosphorylated.
  • cells expressing engineered TSC2 polypeptides that cannot be phosphorylated at position corresponding to a serine residue in the wild-type TSC2 polypeptide sequence, or that cannot be phosphorylated to the full extent as that of a wild- type TSC2 polypeptide having the serine residue.
  • cells provided herein express engineered TSC2 polypeptides in which the serine residue at position S1365 of SEQ ID NO: 5, or a serine residue in a TSC2 polypeptide that corresponds to the serine residue at position S1365 of SEQ ID NO: 5, is substituted with an amino acid with an aliphatic side chain.
  • cells provided herein express engineered TSC2 polypeptides in which the serine residue at position S1364 and/or SI 365 of SEQ ID NO: 5, or a serine residue in a TSC2 polypeptide that corresponds to the serine residue at position S1364 and/or S1365 of SEQ ID NO: 5, is substituted with an amino acid with an aliphatic side chain.
  • cells provided herein express engineered TSC2 polypeptides in which the serine residue at position S1365 of SEQ ID NO: 5, or a serine residue in a polypeptide that corresponds to the serine residue at position S1365 of SEQ ID NO: 5, is substituted with a methionine residue, an alanine residue, a valine residue, a leucine residue, an isoleucine residue, or a phenylalanine residue.
  • cells provided herein express engineered TSC2 polypeptides in which the serine residue at position S1364 and/or S1365 of SEQ ID NO: 5, or a serine residue in a polypeptide that corresponds to the serine residue at position S1364 and/or S1365 of SEQ ID NO: 5, is substituted with a methionine residue, an alanine residue, a valine residue, a leucine residue, an isoleucine residue, or a phenylalanine residue.
  • cells disclosed herein express an engineered TSC2 polypeptide (e.g., SEQ ID NO: 1) having an amino acid substitution at the serine residue at position S1365 of the human TSC2 polypeptide sequence.
  • cells provided herein express engineered TSC2 polypeptides that cannot be phosphorylated to the extent that a non-engineered TSC2 polypeptide having the serine residue(s) can be phosphorylated. In some embodiments, cells provided herein express engineered TSC2 polypeptides that are phosphorylated to an extent that is about 5%,
  • cells provided herein express engineered TSC2 polypeptides that cannot be phosphorylated.
  • engineered TSC2 polypeptides having an amino acid substitution at a residue corresponding to the serine residue at position SI 366, or at both S1365 and S1366, of the mouse TSC2 polypeptide sequence (SEQ ID NO: 6) such that engineered TSC2 polypeptides cannot be phosphorylated at the engineered residue, or cannot be phosphorylated to the extent that a non-engineered TSC2 polypeptide having the serine residue can be phosphorylated.
  • cells provided herein express an engineered TSC2 polypeptide in which the serine residue at position S 1366 of SEQ ID NO: 6, or a serine residue in a TSC2 polypeptide that corresponds to the serine residue at position S 1366 of SEQ ID NO: 6, is substituted with an amino acid with an aliphatic side chain.
  • cells provided herein express an engineered TSC2 polypeptide in which the serine residue at position SI 365 and/or SI 366 of SEQ ID NO: 6, or a serine residue in a TSC2 polypeptide that corresponds to the serine residue at position S1365 and/or S 1366 of SEQ ID NO: 6, is substituted with an amino acid with an aliphatic side chain.
  • cells provided herein express an engineered TSC2 polypeptide in which the serine residue at position S 1366 of SEQ ID NO: 6, or a serine residue in a polypeptide that corresponds to the serine residue at position S1366 of SEQ ID NO: 6, is substituted with a methionine residue, an alanine residue, a valine residue, a leucine residue, an isoleucine residue, or a phenylalanine residue.
  • cells provided herein express an engineered TSC2 polypeptide in which the serine residue at position S1365 and/or S 1366 of SEQ ID NO: 6, or a serine residue in a polypeptide that corresponds to the serine residue at position S1365 and/or S 1366 of SEQ ID NO: 6, is substituted with a methionine residue, an alanine residue, a valine residue, a leucine residue, an isoleucine residue, or a phenylalanine residue.
  • cells provided herein express an engineered TSC2 polypeptide (e.g., SEQ ID NO: 8) having an amino acid substitution at the serine residue at position S 1366 of the mouse TSC2 polypeptide sequence.
  • cells provided herein express an engineered TSC2 polypeptide (e.g., SEQ ID NO: 24) having amino acid substitutions at the serine residue positions S 1365 and SI 366 of the mouse TSC2 polypeptide sequence.
  • cells provided herein express engineered TSC2 polypeptides that cannot be phosphorylated to the extent that a non-engineered TSC2 polypeptide having the serine residue(s) can be
  • cells provided herein express engineered TSC2 polypeptides that are phosphorylated to an extent that is about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or less that a non-engineered TSC2 polypeptide having the serine residue(s). In some embodiments, cells provided herein express engineered TSC2 polypeptides that cannot be phosphorylated.
  • cells provided herein express an engineered TSC2 polypeptide in which the serine residue at position S1367 of SEQ ID NO: 7, or a serine residue in a TSC2 polypeptide that corresponds to the serine residue at position S1367 of SEQ ID NO: 7, is substituted with an amino acid with an aliphatic side chain.
  • cells provided herein express an engineered TSC2 polypeptide in which the serine residue at position SI 366 and/or SI 367 of SEQ ID NO: 7, or a serine residue in a TSC2 polypeptide that corresponds to the serine residue at position S1366 and/or S1367 of SEQ ID NO: 7, is substituted with an amino acid with an aliphatic side chain.
  • cells provided herein express an engineered TSC2 polypeptide in which the serine residue at position S1367 of SEQ ID NO: 7, or a serine residue in a polypeptide that corresponds to the serine residue at position S1367 of SEQ ID NO: 7, is substituted with a methionine residue, an alanine residue, a valine residue, a leucine residue, an isoleucine residue, or a phenylalanine residue.
  • cells provided herein express an engineered TSC2 polypeptide in which the serine residue at position S1366 and/or S1367 of SEQ ID NO: 7, or a serine residue in a polypeptide that corresponds to the serine residue at position S1366 and/or S1367 of SEQ ID NO: 7, is substituted with a methionine residue, an alanine residue, a valine residue, a leucine residue, an isoleucine residue, or a phenylalanine residue.
  • cells provided herein express an engineered TSC2 polypeptide (e.g., SEQ ID NO: 9) having an amino acid substitution at the serine residue at position S1367 of the rat TSC2 polypeptide sequence.
  • cells provided herein express an engineered TSC2 polypeptide (e.g., SEQ ID NO: 26) having amino acid substitutions at the serine residue positions S1366 and SI 367 of the rat TSC2 polypeptide sequence.
  • cells provided herein express engineered TSC2 polypeptides that cannot be phosphorylated to the extent that a non-engineered TSC2 polypeptide having the serine residue(s) can be
  • cells provided herein express engineered TSC2 polypeptides that are phosphorylated to an extent that is about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or less that a non-engineered TSC2 polypeptide having the serine residue(s). In some embodiments, cells provided herein express engineered TSC2 polypeptides that cannot be phosphorylated.
  • cells harboring vectors that include nucleic acid sequences that encode polypeptides that cannot be phosphorylated at a residue corresponding to S1364 and/or S1365 of the human TSC2 polypeptide sequence (SEQ ID NO: 5), S1365 and/or S1366 of the mouse TSC2 polypeptide sequence (SEQ ID NO: 6), or SI 366 and/or SI 367 of the rat TSC2 polypeptides sequence (SEQ ID NO: 7), or that cannot be phosphorylated to the extent that a TSC2 polypeptide having a serine residue at these positions can be phosphorylated, and cells having such vectors.
  • vectors that include nucleic acid sequences that encode polypeptides that cannot be phosphorylated at a residue corresponding to S1364 and/or S1365 of the human TSC2 polypeptide sequence (SEQ ID NO: 5), S1365 and/or S1366 of the mouse TSC2 polypeptide sequence (SEQ ID NO: 6), or SI 366 and/or SI
  • a vector can be introduced into a cell in a transient manner (e.g., maintained as a vector) or in a stable manner (e.g, integrated into the genome).
  • transient manner e.g., maintained as a vector
  • stable manner e.g., integrated into the genome
  • methods and/or techniques that can be used to introduce one or more vectors into a cell include, without limitation, transfection, transduction, electroporation, and infection.
  • nucleic acids encoding engineered TSC2 polypeptides are operably linked to nucleic acids that drive expression of the engineered TSC2 polypeptides in the vectors (e.g., promoter sequences).
  • cells having engineered TSC2 polypeptides that cannot be phosphorylated, or that cannot be phosphorylated to the extent of a wild-type TSC2 polypeptide can have, or can express, endogenous TSC2 proteins.
  • a cell having an engineered TSC2 polypeptide also can have an endogenous wild-type nucleic acid sequence encoding a wild-type TSC2 polypeptide.
  • cells having (e.g., expressing) both: 1) engineered TSC2 polypeptides that cannot be phosphorylated, or that cannot be phosphorylated to the extent of a wild-type TSC2 polypeptide, and 2) an endogenous TSC2 protein the cells exhibit the same or similar activity as a corresponding cell lacking (e.g., not expressing) the endogenous TSC2 protein.
  • cells having engineered TSC2 polypeptides that cannot be phosphorylated, or that cannot be phosphorylated to the extent of a wild-type TSC2 polypeptide do not have, or do not express, endogenous, wild-type TSC2 proteins.
  • a cell having an engineered TSC2 polypeptide can have a genetic alteration in which a wild-type nucleic acid sequence encoding the TSC2 polypeptide has been rendered inactive.
  • the nucleic acid sequence encoding wild-type TSC2 can be modified by any of a variety of genetic manipulation techniques known in the art including, but not limited to, CRISPR-based methods, TALEN-based methods, and other genetic targeting or recombination methods (see, e.g., Roth el ctl, 2018 Nature 559:405-409).
  • a wild-type nucleic acid sequence encoding a TSC2 polypeptide can be rendered inactive by removing, replacing, or mutating a nucleic acid sequence that contributes to expression of the TSC2 polypeptide including, but not limited to, a promoter sequence, an enhancer sequence, a coding sequence of a transcription factor that regulates expression of TSC2, the coding sequence of the TSC2 polypeptide itself, or combinations thereof.
  • a wild-type nucleic acid sequence encoding a TSC2 polypeptide can be rendered inactive via a frameshift caused by one or more modifications or mutations in the nucleic acid sequence encoding the TSC2 polypeptide.
  • cells that can be engineered to include an engineered TSC2 polypeptide that cannot be phosphorylated at a position that corresponds to a wild-type serine residue include immune cells.
  • the immune cells can be CD4+ T cells, CD8+ T cells, Natural Killer cells (NK cells), macrophages, neutrophils, regulatory T cells (Tregs), helper T cells, or any other immune cells and/or inflammatory cells known in the art.
  • CD8+ T cells have been investigated in T cell-based therapies for their role in cellular immune responses. Tumor-specific CD8+ T cells have been found in patients with hematologic malignancies and solid tumors and within the pool of tumor-infiltrating lymphocytes. (Zanetti M, Tapping CD4 T cells for cancer immunotherapy: the choice of personalized genomics, J Immunol. 2015 Mar l;194(5):2049-56). The role of CD8+ T cells has been demonstrated in mouse models of cancer. The presence of CD8+ T cells in tumors has also been shown in human. It has also been shown that the engagement of checkpoint receptors on activated CD8+ T cells represents a major mechanism of tumor- induced immunosuppression.
  • CD8+ T cells have been found to be associated with longer patient survival when tumors display high densities of tertiary lymphoid structures in lung, colorectal, and renal cell cancers (see, e.g., Teng el al., 2015 J Clin Invest. 125:3338-46).
  • CD8+ effector T cells can be generated by stimulating the splenocytes and expanding them in effector promoting conditions with IL-2. Strong IL-2 signaling preferentially skews CD8+ T cells to differentiate into effector T cells. After some time, CD8+ cultures can be processed to remove non-viable cells and assessed for effector function by re-stimulating viable cells with PMA and Ionomycin along with a Golgi blocker to capture cytokines within the cells for later flow cytometry analysis.
  • Interferon gamma IFNg
  • TNFa tumor necrosis factor alpha
  • IL-2 Interleukin-2
  • CD4+ T cells are also known to play a role in adaptive immune responses.
  • CD4+ T cells contribute to anti-tumor immunity through their diverse functions.
  • CD4+ T cells facilitate B cells with isotype switching and affinity maturation.
  • CD4+ T cells are also involved in facilitating the activation and expansion of CD8+ T cells, and the generation and maintenance of memory CD8+ T cells.
  • CD4+ T cells are further involved in tumor protection.
  • activated CD4+ T cells have been found to induce delayed-type hypersensitivity-like reactions and attract inflammatory cells including macrophages, granulocytes, eosinophils, and NK cells in or around the tumor.
  • AT regulatory cell or“Treg cell” refers to a cell that can modulate a T cell response. Regulatory T cells are known for their ability to downregulate the function of other T cells. (Zanetti M, Tapping CD4 T cells for cancer immunotherapy: the choice of personalized genomics, J Immunol. 2015 Mar l;194(5):2049-56). Treg cells express the transcription factor Foxp3, which is not upregulated upon T cell activation and discriminates Tregs from activated effector cells. Tregs are identified by the cell surface markers CD25, CTLA4, and GITR. Several Treg subsets have been identified that have the ability to inhibit autoimmune and chronic inflammatory responses and to maintain immune tolerance in tumor-bearing hosts.
  • IL-10- interleukin 10- secreting T regulatory type 1 (Trl) cells
  • Trl transforming growth factor-b-
  • Th3 T helper type 3
  • Tm CD4+/CD25+ Tregs
  • an immune cell that is engineered to include an engineered TSC2 polypeptide can be a cytotoxic T cell.
  • a cytotoxic T cell can be engineered to express a chimeric antigen receptor (“CAR”) or a T cell receptor (“TCR”).
  • an immune cell that is engineered to include an engineered TSC2 polypeptide can be a B cell.
  • B cells are known to be involved in immune responses and T cell activation.
  • B cells can act as antigen-specific antigen presenting cells.
  • a signal through binding of an antigen to membrane Ig can enhance B cell antigen presentation and T-cell-dependent B cell activation.
  • helper T cell recognition of antigen on the B cell surface the T cell becomes activated and then activates the B cell.
  • an immune cell that is engineered to include an engineered TSC2 polypeptide can be expanded ( e.g ., clonally expanded).
  • an immune cell that is engineered to include an engineered TSC2 polypeptide can be clonally expanded ex vivo (e.g., for use in an adoptive cell therapy).
  • the adoptive cell therapy can be any appropriate adoptive cell therapy.
  • adoptive cell therapies include, without limitation, dendritic cell therapy and synthetic dendritic cell therapy.
  • adoptive cell therapy can include the extraction of tumor infiltrating lymphocytes.
  • increased mTORCl signaling can be determined by any of a variety of techniques or methods known in the art.
  • mTORCl signaling typically results in increased growth, decreased autophagy, and phosphorylation of Ulkl (Unc-51-like kinase-1), p70S6K, and 4EBP1 (elF4E binding protein-1).
  • compositions and methods provided herein can be used to treat cancer.
  • an immune cell that is engineered to include an engineered TSC2 polypeptide (e.g., expressed from a vector introduced into the cell, which vector includes a nucleic acid sequence that encodes the engineered TSC2 polypeptide) that cannot be phosphorylated at a residue corresponding to SI 364 and/or SI 365 of the human TSC2 polypeptide sequence (SEQ ID NO: 5), S1365 and/or S1366 of the mouse TSC2 polypeptide sequence (SEQ ID NO: 6), or S1366 and/or S1367 of the rat TSC2 polypeptides sequence (SEQ ID NO: 7), or that cannot be phosphorylated to the full extent as that of a wild-type TSC2 polypeptide, can be administered to a subject having cancer (e.g., in an adoptive cell therapy) such that the cancer is treated.
  • a subject having cancer e.g., in an adoptive cell therapy
  • the engineered immune cell administered to the subject does not have or express an endogenous, wild type TSC polypeptide. In some embodiments, the engineered immune cell administered to the subject does have or express an endogenous, wild type TSC2 polypeptide.
  • a CD8+ T effector cell that recognizes a cancer cell can be engineered to include a TSC2 polypeptide that cannot be phosphorylated, or that cannot be phosphorylated to the full extent as that of a wild-type TSC2 polypeptide, which CD8+ T effector cell is then administered to a subject that has such a cancer cell.
  • a CD8+ T effector cell that is engineered to include a TSC2 polypeptide that cannot be phosphorylated, or that cannot be phosphorylated to the full extent as that of a wild-type TSC2 polypeptide, is also engineered to express a chimeric antigen receptor or a T cell receptor.
  • an immune cell that is engineered to include an engineered TSC2 polypeptide that cannot be phosphorylated, or that cannot be phosphorylated to the full extent as that of a wild-type TSC2 polypeptide, is more effective in treating cancer in a subject than an immune cell that lacks the engineered TSC2 polypeptide.
  • Cancer types that can be treated include, without limitation, lung cancer (e.g., small cell lung carcinoma or non-small cell lung carcinoma), papillary thyroid cancer, medullary thyroid cancer, differentiated thyroid cancer, recurrent thyroid cancer, refractory differentiated thyroid cancer, lung adenocarcinoma, bronchioles lung cell carcinoma, multiple endocrine neoplasia type 2A or 2B (MEN2A or MEN2B, respectively), pheochromocytoma, parathyroid hyperplasia, breast cancer, colorectal cancer (e.g., metastatic colorectal cancer), papillary renal cell carcinoma,
  • lung cancer e.g., small cell lung carcinoma or non-small cell lung carcinoma
  • papillary thyroid cancer medullary thyroid cancer
  • differentiated thyroid cancer differentiated thyroid cancer
  • recurrent thyroid cancer refractory differentiated thyroid cancer
  • lung adenocarcinoma bronchioles lung cell carcinoma
  • myelodysplastic/myeloproliferative neoplasms myelogenous leukemia, myeloid leukemia, multiple myeloma, myeloproliferative neoplasms, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, non-Hodgkin’s lymphoma, non small cell lung cancer, oral cancer, oral cavity cancer, lip cancer, oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, papillomatosis, paraganglioma, paranasal sinus and nasal cavity cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromosytoma, pituitary cancer, plasma cell neoplasm, pleuropulmonary blastoma, pregnancy and breast cancer, primary central nervous system lymphoma, primary peritoneal cancer, prostate cancer, rectal cancer, renal cell cancer, retinoblastom
  • any of the compositions and methods or methods disclosed provided herein can be used to treat viral diseases.
  • Viral diseases that can be treated include, without limitation, diseases resulting from infection by an adenovirus, a herpesvirus (e.g., HSV-I, HSV-II, CMV, or VZV), a poxvirus (e.g., an orthopoxvirus such as variola or vaccinia, or molluscum contagiosum), a picomavirus (e.g., rhinovirus or enterovirus), an orthomyxovirus (e.g., influenza virus), a paramyxovirus (e.g., parainfluenzavirus, mumps virus, measles virus, and respiratory syncytial virus (RSV)), a coronavirus (e.g., SARS), a papovavirus (e.g., papillomaviruses, such as those that cause genital warts, common warts, or plantar
  • any of the compositions and methods provided herein can be used to treat bacterial diseases.
  • Bacterial diseases that can be treated include, without limitation, diseases resulting from infection by bacteria of, for example, the genus Escherichia, Enterobacter, Salmonella, Staphylococcus, Shigella, Listeria, Aerobacter, Helicobacter, Klebsiella, Proteus, Pseudomonas, Streptococcus, Chlamydia, Mycoplasma, Pneumococcus, Neisseria, Clostridium, Bacillus, Corynebacterium, Mycobacterium, Campylobacter, Vibrio, Serratia, Providencia, Chromobacterium, Brucella, Yersinia, Haemophilus, Bordetella, or Borrelia.
  • any of the compositions and methods provided herein can be used to treat fungal diseases.
  • Fungal diseases that can be treated include, without limitation, candidiasis, aspergillosis, histoplasmosis, and cryptococcal meningitis.
  • any of the compositions and methods provided herein can be used to treat parasitic diseases.
  • Parasitic diseases that can be treated include, without limitation, malaria, pneumocystis camii pneumonia, leishmaniasis, cryptosporidiosis, toxoplasmosis, and trypanosome infection.
  • a disease e.g., any of the variety of cancers, viral diseases, bacterial diseases, fungal diseases, or parasitic diseases disclosed herein
  • a disease e.g., any of the variety of cancers, viral diseases, bacterial diseases, fungal diseases, or parasitic diseases disclosed herein
  • administering one or more agents that result in reduced
  • administration of an agent that results in reduced phosphorylation of TSC2 polypeptides in immune cells results in reduced phosphorylation of a TSC2 polypeptide at a serine residue at position S1365 of the human TSC2 polypeptide sequence (SEQ ID NO: 5).
  • administration of an agent that results in reduced phosphorylation of TSC2 polypeptides in immune cells results in reduced phosphorylation of a TSC2 polypeptide at serine residue positions SI 364 and SI 365 of the human TSC2 polypeptide sequence (SEQ ID NO: 5).
  • administration of an agent that results in reduced phosphorylation of TSC2 polypeptides in immune cells results in reduced phosphorylation of a TSC2 polypeptide at a serine residue at position SI 366 of the mouse TSC2 polypeptide sequence (SEQ ID NO: 6).
  • administration of an agent that results in reduced phosphorylation of TSC2 polypeptides in immune cells results in reduced phosphorylation of a TSC2 polypeptide at serine residue positions SI 365 and SI 366 of the mouse TSC2 polypeptide sequence (SEQ ID NO: 6).
  • administration of an agent that results in reduced phosphorylation of TSC2 polypeptides in immune cells results in reduced phosphorylation of a TSC2 polypeptide at a serine residue at position SI 367 of the rat TSC2 polypeptide sequence (SEQ ID NO: 7).
  • administration of an agent that results in reduced phosphorylation of TSC2 polypeptides in immune cells results in reduced phosphorylation of a TSC2 polypeptide at serine residue at positions SI 366 and SI 367 of the rat TSC2 polypeptide sequence (SEQ ID NO: 7).
  • administration of an agent to a subject that results in reduced phosphorylation of TSC2 polypeptides in immune cells results in a population of TSC2 polypeptides in the immune cell that exhibit decreased
  • TSC2 polypeptides in the cell are not phosphorylated (e.g., a serine residue at position S1364 and/or S1365 of the human TSC2 polypeptide sequence (SEQ ID NO: 5), at a serine
  • TSC2 polypeptides from an immune cell(s) from a subject that has been administered the agent or from an immune cell(s) that has been contacted with the agent in vitro can be isolated (e.g., with a TSC2- specific antibody) and the phosphorylation state of the TSC2 polypeptides can be assayed with a phospho-specific antibody.
  • TSC2- specific antibody e.g., with a TSC2- specific antibody
  • an agent that results in reduced phosphorylation of TSC2 polypeptides in immune cells is a kinase inhibitor.
  • a variety of kinases are known that can be inhibited, including without limitation: AKT1, AKT2, AKT3, CRIK, DMPK1, DMPK2, MRCKa, MRCKb, ROCK1, ROCK2, BARK1, BARK2, GPRK4, GPRK5, GPRK6, GPRK7, RHOK, MAST1, MAST2, MAST3, MAST4, MASTL, LATS1, LATS2, NDR1, NDR2, PDK1, PKACa, PKACb, PKACg, PRKX, PRKY, PKC (e.g., PKCa, PKCb, PKCg, PKCd, PKCt, PKCe, PKCh, PKCi, or PKCz), PKN1, PKN2, PKN3, MSK1, MSK2, p70S6K, p70S6Kb, RSK
  • the agent is a kinase inhibitor (e.g., a kinase inhibitor that inhibits one or more of the kinases disclosed herein).
  • the agent includes two or more kinase inhibitors.
  • the agent includes a kinase inhibitor in combination with at least one other second agent (e.g., a second agent that increases the immune response of an immune cell).
  • a second agent e.g., a second agent that increases the immune response of an immune cell.
  • Non-limiting examples of kinase inhibitors include: MK-5108, palbociclib, capmatinib, rabusertib, SCH-900776, PF-477736, PF-477736, volitinib, crenolanib, pacritinib, adavosertib, afatinib, axitinib, bosutinib, cetuximab, cobimetinib, crizotinib, cabozantinib, dasatinib, entrectinib, erdafitinib, erlotinib, fostamatinib, gefitinib, ibrutinib, imatinib, lapatinib, lenvatinib, mubritinib, nilotinib, pazopanib, pegaptanib, ruxolitinib, sorafenib, sunitin
  • a kinase inhibitor to be administered to a subject to treat a disease inhibits one or more of PKC, p38, MK2 or MK3.
  • kinase inhibitors that inhibit one or more of PKC, p38, MK2 and/or MK3 include: ruboxistaurin, chelerythrine, miy abend C, myricitrin, gossypol, verbascoside, bryostatin 1, pamapimod, PH-797804, BIRB 796, VX-702, SB239063, SB202190, SB203580, SCIO 469, and BMS 582949.
  • a kinase inhibitor can be as described elsewhere (see, e.g., Klaeger et a/., 2017 Science 358: 1148).
  • administration to a subject of an agent that results in reduced phosphorylation of TSC2 polypeptides in immune cells is effective in the treatment of a disease (e.g., any of the variety of cancers, viral diseases, bacterial diseases, fungal diseases, or parasitic diseases disclosed herein).
  • administration to a subject of an agent that results in reduced phosphorylation of TSC2 polypeptides in immune cells results in increased mTORCl signaling in the immune cells such that the immune cells exhibit increased immune activity as compared to a reference immune cell from a reference subject that has not been administered the agent.
  • administration of an agent to a subject that results in reduced phosphorylation of TSC2 polypeptides in immune cells results in reduced
  • TSC2 polypeptides e.g., a population of TSC2 polypeptides in the cell in which the number of TSC2 polypeptides in the population that are phosphorylate is reduced as compared to a population of TSC2 polypeptides in a reference immune cell from a reference subject that has not been administered the agent
  • CD4+ T cells CD8+ T cells
  • NK cells Natural Killer cells
  • macrophages macrophages
  • neutrophils neutrophils
  • Regs regulatory T cells
  • helper T cells or any other immune and inflammatory cells known in the art.
  • administration of an agent to a subject that results in reduced phosphorylation of TSC2 polypeptides in immune cells results in clonal expansion, enhanced synthesis and release of cytokines (e.g., cytokines known in the art to be associated with an increased immune response, including but not limited to TNFa, IFN-g, IFN-a, IFN-b, TGF-b, IL-2, IL-3, IL-4, IL-5, IL-6, IL-10, IL-12, IL-13, and GM- CSF), and other downstream effects known to be associated with an increased immune response.
  • cytokines e.g., cytokines known in the art to be associated with an increased immune response, including but not limited to TNFa, IFN-g, IFN-a, IFN-b, TGF-b, IL-2, IL-3, IL-4, IL-5, IL-6, IL-10, IL-12, IL-13, and GM- CSF
  • a clinical outcome can be determined in a subject that has been administered an agent that results in reduced phosphorylation of TSC2 polypeptides.
  • clinical outcomes include, increased survival (e.g., number of days, months, or years), increased progression-free survival (e.g., number of days, months, or years), increased overall response rate, decreased numbers of cancer cells, decreased tumor burden, and/or decreased numbers of pathogens (e.g., bacteria, viruses, fungi) as compared to a reference subject that has not been administered the agent.
  • An agent e.g., a kinase inhibitor, one or more kinase inhibitors, or a kinase inhibitor in combination with a second agent
  • a subject e.g., a kinase inhibitor, one or more kinase inhibitors, or a kinase inhibitor in combination with a second agent
  • one or more agents can be formulated into a pharmaceutically acceptable composition for administration to a subject having a disease (e.g. , cancer).
  • a therapeutically effective amount of an agent can be formulated together with one or more
  • a pharmaceutical composition can be formulated for administration in solid or liquid form including, without limitation, sterile solutions, suspensions, sustained-release formulations, tablets, capsules, pills, powders, and granules.
  • Pharmaceutically acceptable carriers, fillers, and vehicles that may be used in a pharmaceutical composition described herein include, without limitation, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-poly oxypropylene-block polymers, polyethylene glycol and wool fat.
  • ion exchangers alumina, aluminum stearate, lecithin
  • serum proteins such as human serum albumin
  • buffer substances such as phosphates,
  • a pharmaceutical composition containing one or more agents can be designed for oral or parenteral (including subcutaneous, intramuscular, intravenous, and intradermal) administration.
  • a pharmaceutical composition can be in the form of a pill, tablet, or capsule.
  • Compositions suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions that can contain anti oxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient.
  • the formulations can be presented in unit-dose or multi dose containers, for example, sealed ampules and vials, and may be stored in a freeze dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example, water for injections, immediately prior to use.
  • sterile liquid carrier for example, water for injections, immediately prior to use.
  • Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules, and tablets.
  • a pharmaceutically acceptable composition including one or more agents can be administered locally or systemically.
  • a composition provided herein can be administered locally by injection into tumors.
  • a composition provided herein can be administered systemically, orally, or by injection to a subject (e.g., a human).
  • Effective doses can vary depending on the severity of the disease, the route of administration, the age and general health condition of the subject, excipient usage, the possibility of co-usage with other therapeutic treatments such as use of other agents, and the judgment of the treating physician.
  • an effective amount of a composition containing one or more agents can be any amount that reduces the number of cancer cells present within the subject without producing significant toxicity to the subject.
  • an effective amount of dosage of an agent can be in the range of from about 0.1 mg/kg to about 100 mg/kg of body weight/day, for example, from about 1.0 mg/kg to about 50 mg/kg of body weight/day.
  • the dosage of an agent is in the range of from about 0.1 mg/kg to about 1.0 mg/kg of body weight/day; from about 0.1 mg/kg to about 5 mg/kg of body weight/day; from about 0.1 mg/kg to about 10 mg/kg of body weight/day; from about 0.1 mg/kg to about 25 mg/kg of body weight/day; from about 0.1 mg/kg to about 50 mg/kg of body weight/day; from about 1.0 mg/kg to about 5.0 mg/kg of body weight/day; from about 1.0 mg/kg to about 10 mg/kg of body weight/day; from about 1.0 mg/kg to about 20 mg/kg of body weight/day; from about 1.0 mg/kg to about 25 mg/kg of body weight/day; from about 1.0 mg/kg to about 40 mg/kg of body weight/day; from about 1.0 mg/kg to about 100 mg/kg of body weight/day; from about 10 mg/kg to about 100 mg/kg of body weight/day; from about 25 mg/kg to about 100 mg/
  • the amount of an agent can be increased by, for example, two fold. After receiving this higher amount, the subject can be monitored for both responsiveness to the treatment and toxicity symptoms, and adjustments made accordingly.
  • the effective amount can remain constant or can be adjusted as a sliding scale or variable dose depending on the subject’s response to treatment.
  • Various factors can influence the actual effective amount used for a particular application. For example, the frequency of administration, duration of treatment, use of multiple treatment agents, route of administration, and severity of the condition ( e.g ., cancer) may require an increase or decrease in the actual effective amount administered.
  • the frequency of administration of an agent can be any amount that reduces the number of cancer cells present within the subject without producing significant toxicity to the subject.
  • the frequency of administration of an agent can be from about two to about three times a week to about two to about three times a month.
  • the frequency of administration of an agent can remain constant or can be variable during the duration of treatment.
  • a course of treatment with a composition containing an agent can include rest periods.
  • a composition containing one or more agents can be administered daily over a two week period followed by a two week rest period, and such a regimen can be repeated multiple times.
  • the effective amount various factors can influence the actual frequency of administration used for a particular application.
  • the effective amount, duration of treatment, use of multiple treatment agents, route of administration, and severity of the condition may require an increase or decrease in administration frequency.
  • An effective duration for administering a composition containing one or more agents can be any duration that reduces the severity of the condition (e.g., the number of cancer cells present within the subject) without producing significant toxicity to the subject.
  • the effective duration can vary from several days to several weeks. In general, the effective duration can range in duration from about one week to about four weeks. Multiple factors can influence the actual effective duration used for a particular treatment. For example, an effective duration can vary with the frequency of administration, effective amount, use of multiple treatment agents, route of
  • an agent can be contacted with an immune cell in vitro for any of a variety of purposes including, without limitation, testing specific cell types, testing the effective, maximum, and/or minimum dosage of the agent, and/or testing duration of contact of the agent.
  • a test agent can be contacted with an immune cell in vitro to determine whether it is effective. Any suitable immune cell can be contacted in vitro with the agent including, without limitation, CD4+ T cells,
  • the in vitro agent is a kinase inhibitor.
  • the in vitro agent includes two or more kinase inhibitors.
  • the in vitro agent includes a kinase inhibitor in combination with at least one other second agent (e.g., a second agent that increases the immune response of an immune cell).
  • Effectiveness of the in vitro agent(s) on stimulating the immune cell can be assessed by any of a variety of techniques known in the art.
  • clonal expansion is assessed.
  • enhanced synthesis and release of cytokines e.g., cytokines known in the art to be associated with an increased immune response, including but not limited to TNFa, IFN-g, TGF-b, IL-4, IL-10, IL-13
  • cytokines e.g., cytokines known in the art to be associated with an increased immune response, including but not limited to TNFa, IFN-g, TGF-b, IL-4, IL-10, IL-13
  • engineered TSC2 polypeptides that act as if they are constitutively phosphorylated. Such engineered TSC2 polypeptides can be considered to be“pseudo-phosphorylated”.
  • pseudo- phosphorylated TSC2 polypeptides having an amino acid substitution at a residue corresponding to the serine residue at position S1365 of the human TSC2 polypeptide sequence (SEQ ID NO: 5).
  • pseudo- phosphorylated TSC2 polypeptides having an amino acid substitution at a residue corresponding to the serine residue at position S1364 and/or S1365 of the human TSC2 polypeptide sequence SEQ ID NO: 5).
  • the serine residue at position S1365 of SEQ ID NO: 5, or a serine residue in a polypeptide that corresponds to the serine residue at position S1365 of SEQ ID NO: 5, is substituted with an aspartic acid or a glutamic acid residue.
  • the serine residue at position SI 364 and/or SI 365 of SEQ ID NO: 5, or a serine residue in a polypeptide that corresponds to the serine residue at position S1364 and/or S1365 of SEQ ID NO: 5, is substituted with an aspartic acid or a glutamic acid residue.
  • a pseudo-phosphorylated TSC2 polypeptide having a glutamic acid substitution at the serine residue at position S1365 of the human TSC2 polypeptide sequence is provided (e.g., SEQ ID NO: 2).
  • a pseudo-phosphorylated TSC2 polypeptide having glutamic acid substitutions at the serine residue positions S1364 and S1365 of the human TSC2 polypeptide sequence is provided (e.g., SEQ ID NO: 21).
  • TSC2 polypeptides having an amino acid substitution at a residue corresponding to the serine residue at position SI 366, or at both SI 365 and SI 366, of the mouse TSC2 polypeptide sequence (SEQ ID NO: 6) such that engineered TSC2 polypeptides act as if they are constitutively phosphorylated.
  • the serine residue at position SI 366 of SEQ ID NO: 6, or a serine residue in a polypeptide that corresponds to the serine residue at position SI 366 of SEQ ID NO: 6, is substituted with an aspartic acid or a glutamic acid residue.
  • the serine residue at position S1365 and/or S1366 of SEQ ID NO: 6, or a serine residue in a polypeptide that corresponds to the serine residue at position S1365 and/or SI 366 of SEQ ID NO: 6, is substituted with an aspartic acid or a glutamic acid residue.
  • a pseudo-phosphorylated TSC2 polypeptide having an amino acid substitution at the serine residue at position SI 366 of the mouse TSC2 polypeptide sequence is provided (e.g., SEQ ID NO: 10).
  • a pseudo-phosphorylated TSC2 polypeptide having amino acid substitutions at the serine residue positions SI 365 and SI 366 of the mouse TSC2 polypeptide sequence is provided (e.g., SEQ ID NO: 25).
  • TSC2 polypeptides having an amino acid substitution at a residue corresponding to the serine residue at position SI 367, or at both S1366 and S1367, of the rat TSC2 polypeptide sequence (SEQ ID NO: 7) such that engineered TSC2 polypeptides act as if they are constitutively phosphorylated.
  • the serine residue at position S1367 of SEQ ID NO: 7, or a serine residue in a polypeptide that corresponds to the serine residue at position SI 367 of SEQ ID NO: 7, is substituted with an aspartic acid or a glutamic acid residue.
  • the serine residue at position S1366 and/or S1367 of SEQ ID NO: 7, or a serine residue in a polypeptide that corresponds to the serine residue at position S1366 and/or SI 367 of SEQ ID NO: 7, is substituted with an aspartic acid or a glutamic acid residue.
  • a pseudo-phosphorylated TSC2 polypeptide having an amino acid substitution at the serine residue at position SI 367 of the rat TSC2 polypeptide sequence is provided (e.g., SEQ ID NO: 11).
  • a pseudo-phosphorylated TSC2 polypeptide having amino acid substitutions at the serine residue at positions S1366 and S1367 of the rat TSC2 polypeptide sequence is provided (e.g, SEQ ID NO: 27).
  • pseudo-phosphorylated TSC2 polypeptides exhibit increased activity in their ability to down-regulate the mTORCl pathway as compared to wild-type TSC2 polypeptides that are not phosphorylated at the engineered amino acid position(s).
  • vectors that include nucleic acid sequences that encode polypeptides that act as if they are constitutively phosphorylated at a residue
  • a vector that includes nucleic acid sequences that encode polypeptides that act as if they are constitutively phosphorylated at a residue corresponding to S1364 and/or S1365 of the human TSC2 polypeptide sequence (SEQ ID NO: 5), S1365 and/or S1366 of the mouse TSC2 polypeptide sequence (SEQ ID NO: 6), or SI 366 and/or SI 367 of the rat TSC2 polypeptides sequence (SEQ ID NO: 7) can be any appropriate type of vector.
  • vectors include, without limitation, plasmids (e.g., expression plasmids) and vectors (e.g., viral vectors such as lentiviral vectors, retroviral vectors, adenovirus vectors, and adeno-associated virus vectors).
  • plasmids e.g., expression plasmids
  • vectors e.g., viral vectors such as lentiviral vectors, retroviral vectors, adenovirus vectors, and adeno-associated virus vectors.
  • a vector that includes nucleic acid sequences that encode polypeptides that act as if they are constitutively phosphorylated at a residue corresponding to SI 364 and/or S1365 of the human TSC2 polypeptide sequence (SEQ ID NO: 5), S1365 and/or S1366 of the mouse TSC2 polypeptide sequence (SEQ ID NO: 6), or SI 366 and/or SI 367 of the rat TSC2 polypeptides sequence (SEQ ID NO: 7) can be a lentiviral vector.
  • a vector described herein also includes a promoter
  • the promoter can operably linked to a nucleic acid sequence that encodes polypeptides that act as if they are constitutively phosphorylated at a residue corresponding to S1364 and/or S1365 of the human TSC2 polypeptide sequence (SEQ ID NO: 5), S1365 and/or S1366 of the mouse TSC2 polypeptide sequence (SEQ ID NO: 6), or S1366 and/or S1367 of the rat TSC2 polypeptides sequence (SEQ ID NO: 7) (e.g., such that the promoter can drive expression of the nucleic acid sequence).
  • a promoter can be any appropriate promoter.
  • a promoter can be a constitutive promoter.
  • a promoter can be a viral promoter.
  • a promoter can be an inducible promoter.
  • a promoter can be a cell-specific and/or tissue-specific promoter.
  • promoters that can be used to drive expression of nucleic acid sequences that encode polypeptides that act as if they are constitutively phosphorylated at a residue corresponding to SI 364 and/or S1365 of the human TSC2 polypeptide sequence (SEQ ID NO: 5), S1365 and/or S1366 of the mouse TSC2 polypeptide sequence (SEQ ID NO: 6), or S1366 and/or S1367 of the rat TSC2 polypeptides sequence (SEQ ID NO: 7) include, without limitation, CMV.
  • a promoter can be as described elsewhere (see, e.g., Morgan el al, 2016 Biomedicines. 4:9).
  • cells having vectors that include nucleic acid sequences that encode polypeptides that act as if they are constitutively phosphorylated at a serine residue in any of the polypeptides described herein do not express endogenous, wild-type TSC2.
  • the nucleic acid sequence encoding wild-type TSC2 can be modified by any of a variety of genetic manipulation techniques known in the art including, but not limited to, CRISPR-based methods, TALEN-based methods, and other genetic targeting or recombination methods (see, e.g., Roth et al., 2018 Nature 559:405-409).
  • constitutively phosphorylated at a position that corresponds to a wild-type serine residue in any of the polypeptides described herein do express endogenous, wild-type TSC2.
  • cells having both: 1) vectors that include nucleic acid sequences that encode engineered TSC2 polypeptides that act as if they are constitutively phosphorylated, and 2) a nucleic acid sequence encoding an endogenous TSC2 protein the cells exhibit the same or similar activity as a corresponding cell lacking the nucleic acid sequence encoding the endogenous TSC2 protein.
  • TSC2 polypeptides disclosed herein can be engineered at SI 365 of the human TSC2 polypeptide sequence (SEQ ID NO: 5), S1366 of the mouse TSC2 polypeptide sequence (SEQ ID NO: 6), or SI 367 of the rat TSC2 polypeptide sequence (SEQ ID NO: 7) with any mutation or modification that results in TSC2 mimicking constitutive phosphorylation at the respective positions in human (SI 365), mouse (SI 366), or rat (S1367).
  • TSC2 polypeptides disclosed herein can be engineered at S1364 and S1365 of the human TSC2 polypeptide sequence (SEQ ID NO: 5), S1365 and S1366 of the mouse TSC2 polypeptide sequence (SEQ ID NO: 6), or S1366 and S1367 of the rat TSC2 polypeptide sequence (SEQ ID NO: 7) with any mutation or modification that results in TSC2 mimicking constitutive phosphorylation at the respective positions in human (S1364 and S1365), mouse (S1365 and S1366), or rat (S1366 and S1367).
  • TSC2 polypeptides from other species can be engineered with any mutation or modification (e.g., a residue corresponding to human SI 364 and/or S1365, mouse S1365 and/or S1366, and/or rat S1366 and/or S1367) that results in TSC2 mimicking constitutive phosphorylation.
  • any mutation or modification e.g., a residue corresponding to human SI 364 and/or S1365, mouse S1365 and/or S1366, and/or rat S1366 and/or S1367
  • nucleic acids encoding engineered TSC2 polypeptides that act as if they are constitutively phosphorylated at a position corresponding to a serine residue in a wild-type TSC2 polypeptide sequence.
  • nucleic acids provided herein encode pseudo-phosphorylated TSC2 polypeptides that exhibit increased activity in their ability to down-regulate the mTORCl pathway as compared to wild-type TSC2 polypeptides that are not phosphorylated at the engineered position.
  • nucleic acids provided herein encode pseudo-phosphorylated TSC2 polypeptides in which the serine residue at position S1365 of SEQ ID NO: 5, position S1366 of SEQ ID NO: 6, or position 1367 of SEQ ID NO: 7, or a serine residue in a TSC2 polypeptide that corresponds to the serine residues at those positions, is substituted with an aspartic acid or a glutamic acid residue.
  • nucleic acids provided herein encode pseudo-phosphorylated TSC2 polypeptides in which the serine residue at positions S1364 and/or S1365 of SEQ ID NO: 5, positions S1365 and/or S1366 of SEQ ID NO: 6, or positions S1366 and/or S1367 of SEQ ID NO: 7, or a serine residue in a TSC2 polypeptide that corresponds to the serine residues at those positions, is substituted with an aspartic acid or a glutamic acid residue.
  • nucleic acids provided herein include the genetic codons GAA or GAG at positions that are translated to a glutamic acid residue at positions S1364 and/or SI 365 of the human TSC2 polypeptide sequence (SEQ ID NO: 5), positions S1365 and/or S1366 of the mouse TSC2 polypeptide sequence (SEQ ID NO: 6), or positions S1366 and/or S1367 of the rat TSC2 polypeptide sequence (SEQ ID NO: 7).
  • nucleic acids provided herein include the genetic codons GAT or GAC at positions that are translated to an aspartic acid residue at positions S1364 and/or S1365 of the human TSC2 polypeptide sequence (SEQ ID NO: 5), positions SI 365 and/or SI 366 of the mouse TSC2 polypeptide sequence (SEQ ID NO: 6), or positions S1366 and/or S1367 of the rat TSC2 polypeptide sequence (SEQ ID NO: 7).
  • a nucleic acid sequence encoding an engineered TSC2 polypeptide having a glutamic acid substitution at the serine residue at position S1365 of the human TSC2 polypeptide sequence is provided (e.g., a nucleic acid sequence of SEQ ID NO: 4).
  • a nucleic acid sequence encoding an engineered TSC2 polypeptide having glutamic acid substitutions at the serine residue positions S1364 and S1365 of the human TSC2 polypeptide sequence is provided (e.g., a nucleic acid sequence of SEQ ID NO: 23).
  • cells expressing engineered TSC2 polypeptides that act as if they are constitutively phosphorylated at a position corresponding to a serine residue in the wild-type TSC2 polypeptide sequence.
  • cells provided herein include pseudo-phosphorylated TSC2 polypeptides that exhibit increased activity in their ability to down-regulate the mTORCl pathway as compared to wild-type TSC2 polypeptides that are not phosphorylated at the engineered position.
  • cells provided herein express pseudo-phosphorylated TSC2 polypeptides in which the serine residue at position SI 365 of SEQ ID NO: 5, or a serine residue in a polypeptide that corresponds to the serine residue at position S1365 of SEQ ID NO: 5, is substituted with an aspartic acid or a glutamic acid residue.
  • cells provided herein express pseudo-phosphorylated TSC2 polypeptides in which the serine residue positions S1364 and S1365 of SEQ ID NO: 5, or a serine residue in a polypeptide that corresponds to the serine residue at positions SI 364 and SI 365 of SEQ ID NO: 5, are substituted with an aspartic acid or a glutamic acid residue.
  • cells expressing pseudo-phosphorylated TSC2 polypeptides having a glutamic acid substitution at a residue corresponding to the serine residue at position S1365 of the human TSC2 polypeptide sequence e.g., SEQ ID NO: 2.
  • cells provided herein express an engineered TSC2 polypeptide having glutamic acid substitutions at the serine residue positions S1364 and S1365 of the human TSC2 polypeptide sequence (e.g., SEQ ID NO: 21).
  • cells expressing pseudo-phosphorylated TSC2 polypeptides having an amino acid substitution at a residue corresponding to the serine residue at position SI 366, or both SI 365 and S1366, of the mouse TSC2 polypeptide sequence (SEQ ID NO: 6) such that engineered TSC2 polypeptides act as if they are constitutively phosphorylated at the serine residue.
  • the cells provided herein express a pseudo-phosphorylated TSC2 polypeptide in which the serine residue at position S1366 of SEQ ID NO: 6, or a serine residue in a polypeptide that corresponds to the serine residue at position S1366 of SEQ ID NO: 6, is substituted with an aspartic acid or a glutamic acid residue.
  • the cells provided herein express a pseudo-phosphorylated TSC2 polypeptide in which the serine residue at positions S1365 and S1366 of SEQ ID NO: 6, or a serine residue in a polypeptide that corresponds to the serine residue at positions S1365 and S1366 of SEQ ID NO: 6, are substituted with an aspartic acid or a glutamic acid residue.
  • cells provided herein express a pseudo-phosphorylated TSC2 polypeptide having a glutamic acid substitution at the serine residue at position S1366 of the mouse TSC2 polypeptide sequence (e.g., SEQ ID NO: 10).
  • cells provided herein express an engineered TSC2 polypeptide having glutamic acid substitutions at the serine residue positions S1366 and S1366 of the mouse TSC2 polypeptide sequence (e.g., SEQ ID NO: 25).
  • cells provided herein express a pseudo-phosphorylated TSC2 polypeptide in which the serine residue at position S1367 of SEQ ID NO: 7, or a serine residue in a polypeptide that corresponds to the serine residue at position S1367 of SEQ ID NO: 7, is substituted with a aspartic acid or a glutamic acid residue.
  • cells provided herein express a pseudo-phosphorylated TSC2 polypeptide in which the serine residue at positions SI 366 and SI 367 of SEQ ID NO: 7, or a serine residue in a polypeptide that corresponds to the serine residue at positions SI 366 and SI 367 of SEQ ID NO: 7, are substituted with a aspartic acid or a glutamic acid residue.
  • cells provided herein express an engineered TSC2 polypeptide having a glutamic acid substitution at the serine residue at position S1367 of the rat TSC2 polypeptide sequence (e.g., SEQ ID NO: 11).
  • cells provided herein express an engineered TSC2 polypeptide having glutamic acid substitutions at the serine residue positions S1366 and S1367 of the rat TSC2 polypeptide sequence (e.g., SEQ ID NO: 27).
  • cells harboring vectors that include nucleic acids that encode polypeptides that act as if they are constitutively phosphorylated at a serine residue corresponding to S1365, or both S1364 and S1365, of the human TSC2 polypeptide sequence (SEQ ID NO: 5), S1366, or both S1365 and S1366, of the mouse TSC2 polypeptide sequence (SEQ ID NO: 6), or S1367, or both S1366 and S1367, of the rat TSC2 polypeptides sequence (SEQ ID NO: 7).
  • a vector that includes nucleic acid sequences that encode polypeptides that act as if they are constitutively phosphorylated at a residue corresponding to S1365, or both S1364 and S1365, of the human TSC2 polypeptide sequence (SEQ ID NO: 5), S1366, or both S1365 and S1366, of the mouse TSC2 polypeptide sequence (SEQ ID NO: 6), or S1367, or both S1366 and S1367, of the rat TSC2 polypeptides sequence (SEQ ID NO: 7) can be introduced into a cell using any appropriate methods and/or techniques.
  • a vector can be introduced into a cell in a transient manner (e.g., maintained as a vector) or in a stable manner (e.g., integrated into the genome).
  • transient manner e.g., maintained as a vector
  • stable manner e.g., integrated into the genome
  • methods and/or techniques that can be used to introduce one or more vectors into a cell include, without limitation, transfection, transduction, electroporation, and infection.
  • nucleic acids encoding pseudo- phosphorylated TSC2 polypeptides are operably linked to nucleic acids that drive expression of pseudo-phosphorylated TSC2 polypeptides in the vectors (e.g., promoter sequences).
  • cells having pseudo-phosphorylated TSC2 polypeptides that act as if they are constitutively phosphorylated can have, or can express, endogenous, TSC2 proteins.
  • a cell having a pseudo-phosphorylated TSC2 polypeptide also can have an endogenous wild-type nucleic acid sequence encoding a wild-type TSC2 polypeptide.
  • the cells having (e.g., expressing) both: 1) engineered pseudo-phosphorylated TSC2 polypeptides that act as if they are constitutively phosphorylated, and 2) an endogenous TSC2 protein the cells exhibit the same or similar activity as a corresponding cell lacking (e.g., not expressing) the endogenous TSC2 protein.
  • cells having pseudo-phosphorylated TSC2 polypeptides that act as if they are constitutively phosphorylated do not have, or do not express, endogenous, wild-type TSC2 proteins.
  • a cell having a pseudo- phosphorylated TSC2 polypeptide can have a genetic alteration in which a wild-type nucleic acid sequence encoding the TSC2 polypeptide has been rendered inactive.
  • the nucleic acid sequence encoding wild-type TSC2 can be modified by any of a variety of genetic manipulation techniques known in the art including, but not limited to, CRISPR-based methods, TALEN-based methods, and other genetic targeting or recombination methods (see, e.g., Roth et al., 2018 Nature 559:405-409).
  • a wild-type nucleic acid sequence encoding a TSC2 polypeptide can be rendered inactive by removing, replacing, or mutating a nucleic acid sequence that contributes to expression of the TSC2 polypeptide including, but not limited to, a promoter sequence, an enhancer sequence, a coding sequence of a transcription factor that regulates expression of TSC2, the coding sequence of the TSC2 polypeptide itself, or combinations thereof.
  • a wild-type nucleic acid sequence encoding a TSC2 polypeptide can be rendered inactive via a frameshift caused by one or more modifications or mutations in the nucleic acid sequence encoding the TSC2 polypeptide.
  • cells that can be engineered to include a pseudo- phosphorylated TSC2 polypeptide include immune cells.
  • the immune cells can be CD4+ T cells, CD8+ T cells, Natural Killer cells (NK cells), macrophages, neutrophils, regulatory T cells (Tregs), helper T cells, B cells, or any other immune cells and/or inflammatory cells known in the art. Relevant aspects of certain of these cells are disclosed elsewhere herein.
  • an immune cell that is engineered to include an engineered TSC2 polypeptide can be a cytotoxic T cell.
  • a cytotoxic T cell can be engineered to express a CAR or a TCR.
  • an immune cell that is engineered to include an engineered TSC2 polypeptide can be expanded (e.g., clonally expanded).
  • an immune cell that is engineered to include an engineered TSC2 polypeptide can be clonally expanded ex vivo (e.g., for use in an adoptive cell therapy).
  • the adoptive cell therapy can be any appropriate adoptive cell therapy.
  • adoptive cell therapies include, without limitation, dendritic cell therapy and synthetic dendritic cell therapy.
  • adoptive cell therapy can include the extraction of tumor infiltrating lymphocytes.
  • decreased mTORCl signaling can be determined by any of a variety of techniques or methods known in the art. For example, inhibited mTORCl signaling typically results in decreased growth, increased autophagy, and less
  • Ulkl Unc-51-like kinase-1
  • p70S6K ribosomal protein S6 kinase
  • 4EBP1 elF4E binding protein- 1.
  • compositions and methods provided herein can be used to treat cancer.
  • an immune cell that is engineered to include an engineered TSC2 polypeptide (e.g., expressed from a vector introduced into the cell, which vector includes a nucleic acid sequence that encodes the engineered TSC2 polypeptide) that acts as if it is constitutively phosphorylated at a residue corresponding to SI 365, or both S1364 and S1365, of the human TSC2 polypeptide sequence (SEQ ID NO: 5), S1366, or both S1365 and S1366, of the mouse TSC2 polypeptide sequence (SEQ ID NO: 6), or S1367, or both S1366 and S1367, of the rat TSC2 polypeptides sequence (SEQ ID NO: 7) can be administered to a subject having cancer (e.g., in an adoptive cell therapy) such that the cancer is treated.
  • an engineered TSC2 polypeptide e.g., expressed from a vector introduced into the cell, which vector includes a nucle
  • the engineered immune cell administered to the subject does not have or express an endogenous, wild type TSC polypeptide. In some embodiments, the engineered immune cell administered to the subject does have or express an endogenous, wild type TSC2 polypeptide.
  • a CD8+ T effector cell that recognizes a cancer cell can be engineered to include a TSC2 polypeptide that acts as if it is constitutively non-phosphorylated, which CD8+ T effector cell is then administered to a subject that has such a cancer cell. This enhances mTORCl activation with stimulation to increase effector function.
  • a CD8+ T effector cell that is engineered to include a TSC2 polypeptide acts as if it is constitutively non-phosphorylated is also engineered to express a chimeric antigen receptor or a T cell receptor.
  • an immune cell that is engineered to include an engineered TSC2 polypeptide that acts as if it is constitutively phosphorylated is more effective in treating cancer in a subject than an immune cell that lacks the engineered TSC2 polypeptide.
  • Cancer types that can be treated include, without limitation, lung cancer (e.g., small cell lung carcinoma or non-small cell lung carcinoma), papillary thyroid cancer, medullary thyroid cancer, differentiated thyroid cancer, recurrent thyroid cancer, refractory differentiated thyroid cancer, lung adenocarcinoma, bronchioles lung cell carcinoma, MEN2A, MEN2B, pheochromocytoma, parathyroid hyperplasia, breast cancer, colorectal cancer (e.g., metastatic colorectal cancer), papillary renal cell carcinoma, ganglioneuromatosis of the gastroenteric mucosa, inflammatory
  • myofibroblastic tumor or cervical cancer, ALL, AML, cancer in adolescents, adrenal cancer, adrenocortical carcinoma, anal cancer, appendix cancer, astrocytoma, atypical teratoid/rhabdoid tumor, basal cell carcinoma, bile duct cancer, bladder cancer, bone cancer, brain stem glioma, brain tumor, breast cancer, bronchial tumor, Burkitt lymphoma, carcinoid tumor, unknown primary carcinoma, cardiac tumors, cervical cancer, childhood cancers, chordoma, CLL, CML, chronic myeloproliferative neoplasms, colon cancer, colorectal cancer, craniopharyngioma, cutaneous T-cell lymphoma, bile duct cancer, ductal carcinoma in situ, embryonal tumors, endometrial cancer, ependymoma, esophageal cancer, esthesioneuroblastoma, Ewing sarcoma, extracranial germ cell tumor,
  • compositions and methods provided herein can be are useful for other situations and/or can be used to treat other diseases.
  • examples of other situations where the compositions and methods provided herein can be useful include, without limitation, situations of tissue, skin, and organ transplantation.
  • examples of other diseases that the compositions and methods can be used to treat include, without limitation, graft-versus-host disease (GVHD), allergies, asthma, autoimmune diseases (such as systemic lupus erythematosus and rheumatoid arthritis), multiple sclerosis, and inflammatory bowel disease.
  • GVHD graft-versus-host disease
  • allergies asthma
  • autoimmune diseases such as systemic lupus erythematosus and rheumatoid arthritis
  • multiple sclerosis inflammatory bowel disease.
  • cells comprising a pseudo- phosphorylated TSC2 polypeptide can be administered to patients in need (e.g., in an adoptive cell therapy) resulting in decreased mTORCl activities and thus treating diseases including situations of tissue, skin and organ transplantation, in GVHD, or allergies, or in autoimmune diseases such as systemic lupus erythematosus and multiple sclerosis.
  • methods of generating a persistent T cell in a subject include administering to a subject (e.g., in an adoptive cell therapy) an engineered immune cell comprising a vector.
  • the vector comprises a nucleic acid encoding a polypeptide comprising SEQ ID NO: 2 that is operably linked to a nucleic acid that drives expression of the polypeptide in the engineered immune cell.
  • the engineered immune cell recognizes an antigen. In some embodiments, upon recognizing the antigen, the engineered immune cell exhibits decreased effector cell activity as compared to a reference T cell that lacks the vector. In some embodiments, upon recognizing the antigen, the engineered immune cell exhibits long-term persistence and memory activity as compared to a reference T cell that lacks the vector. In some embodiments, upon recognizing the antigen, the engineered immune cell becomes a persistent T cell.
  • the decreased effector activity of the engineered immune cell can include a decline in mTORCl signaling.
  • the engineered immune cell is derived from an endogenous immune cell obtained from the subject.
  • the phrase“derived from” means that the endogenous immune cell is obtained from the subject, after which it is modified (e.g., via introduction of a vector having an nucleic acid sequence encoding a modified TSC2 polypeptide as described herein) to generate the engineered immune cell.
  • the engineered immune cell is a CD 8+ T cell.
  • the persistent T cell is a memory T cell.
  • the CD8+ T cell is further engineered to express a CAR or a TCR.
  • the engineered immune cell is a regulatory T cell.
  • the persistent T cell is a persistent T regulatory cell.
  • methods of generating a persistent T cell in vitro include providing an immune cell, introducing into the immune cell a vector thereby generating an engineered immune cell, contacting the engineered immune cell with an antigen that is recognized by the engineered immune cell, and culturing the engineered immune cell under conditions and for a time sufficient such that the engineered immune cell becomes the persistent T cell.
  • the vector comprises a nucleic acid encoding a polypeptide comprising SEQ ID NO: 2 that is operably linked to a nucleic acid that drives expression of the polypeptide in the immune cell.
  • the engineered immune cell exhibits decreased mTORCl signaling as compared to a reference immune cell that does not comprise the vector.
  • the engineered immune cell expresses one or more polypeptides that contain a targeted pseudophosphorylation modification (e.g., that can result in decreased mTORCl signaling) as compared to a reference immune cell that does not comprise the vector.
  • the immune cell is a CD 8+ T cell, and the generated persistent T cell is a memory T cell.
  • the CD8+ T cell is further engineered to express a chimeric antigen receptor or a T cell receptor.
  • a persistent T cell generated in vitro is administered to a subject (e.g., in an adoptive cell therapy).
  • the subject exhibits a disease.
  • administration of the persistent T cell to the subject treats the disease.
  • the disease is cancer, a viral disease, a bacterial disease, fungal disease, or a parasitic disease (e.g., any of the cancers, viral diseases, bacterial diseases, fungal diseases, or parasitic diseases disclosed herein).
  • the immune cell is obtained from the subject to be treated (e.g., an autologous cell). In some embodiments, the immune cell is obtained from a subject other than the subject to be treated (e.g. , an allogenic cell). In some
  • the immune cell is a regulatory T cell, and the persistent T cell is a persistent T regulatory cell.
  • the persistent T regulatory cell is administered to a subject (e.g., in an adoptive cell therapy).
  • the subject exhibits a disease.
  • administration of the persistent T regulatory cell to the subject treats the disease.
  • the disease is asthma, an autoimmune disease, or graft vs. host disease.
  • the immune cell is obtained from the subject (e.g., any of the cancers, viral diseases, bacterial diseases, fungal diseases, or parasitic diseases disclosed herein).
  • Example 1 Substitution of murine TSC2 SI 366 (human TSC2 SI 365) modulates mTORCl signaling
  • NRCMs Neonatal rat cardiomyocyte studies
  • NRCMs are freshly isolated from pregnant female rates, cells isolated and cultured for 24 hours in DMEM with 10% FBS and antibiotics prior to study, as described elsewhere (see, e.g., Lee et al, 2015 Nature 519:472-476). Cells were also transfected with plasmids expressing human TSC2 polypeptides: TSC2-WT, TSC2-S1364A, TSC2- S1364E, TSC2-S1365A, of TSC2-S1365E performed with Takara Clontech Xfect reagent per manufacturer protocol.
  • cDNA underwent PCR amplification using TaqMan probes for brain or B-type natriuretic peptide (BNP) (mouse # Mm() 1255770_g 1. rat #Rn00580641_ml), and glyceraldehyde- 3-phosphate dehydrogenase (GAPDH) (mouse #99999915_g 1. rat #Rn() 1775763_g 1 ) (Applied Biosystems). The threshold cycle value was determined using the crossing point method. Samples were normalized to the GAPDH value for each run.
  • NRCMs were infected with an adenovirus (10 MO I) expressing a tandem fluorescent (GFP-RFP) tagged LC3 15 .
  • This expresses LC3 with both green and red fluorescence as the autophagosomal membrane is forming; but upon merging with the acidic lysosome (autolysosome), the GFP signal is quenched, leaving RFP. The rise in RFP provides a marker of autophagic flux.
  • myocytes were further transfected with plasmid encoding for WT-TSC2, or SA or SE mutant TSC2, and further stimulated for 48 hours with endothelin 1 (10 nM). Dot counts for both colors/cell were determined using Image J software (Ver 1.52a, NIH).
  • PKG activity was assessed by in vitro colorimetric assay (Cyclex, Cat #CY-1161, Nagano, Japan) following the manufacturer’s instructions.
  • the assay provides cGMP substrate, and a kinase-specific peptide-target to assess phosphorylation activity.
  • NRVMs were infected with an adenovirus (10 MOI) expressing a tandem fluorescent (GFP-RFP) tagged LC315 (LC3-GFP-RFP).
  • This adenovirus expresses LC3 with both green and red fluorescence as the autophagosomal membrane is forming; but upon merging with the acidic lysosome (autolysosome), the GFP signal is quenched, leaving RFP.
  • the rise in RFP provides a marker of autophagic flux.
  • NRVMs were then stimulated with endothelin- 1 (ET1), and in turn treated with either a protein kinase G activator (cGMP) or inhibitor (DT3).
  • ET1 stimulates myocyte growth (hypertrophy) and this is markedly reduced by protein kinase G stimulation.
  • cGMP protein kinase G activator
  • DT3 inhibitor
  • ET1 stimulates myocyte growth (hypertrophy) and this is markedly reduced by protein kinase G stimulation.
  • These cells display an increase in red dots indicating enhanced autophagy.
  • inhibiting PKG increases cell size and markedly reduces autophagy as reflected by the fall in both green and red dots ( Figure 1).
  • a serine duplet is present in TSC2 numbered as residues SI 364 and S1365 in the human TSC2 sequence set forth in SEQ ID NO: 5. These sites are all highly homologous between human and other mammalian species, though the numbering is slightly different for the T1271 AMPK site (mouse it is T1270), and for all sites identified at and above SI 364 (mouse number would be one higher for each).
  • residues S1364 and S1365 in the human TSC2 sequence set forth in SEQ ID NO: 5 would be numbered a S1365 and S1366 in the mouse TSC2 sequence set forth in SEQ ID NO: 6.
  • TSC2 when modifying either the first serine in the doublet (hsS1364, mmS1364) or the second serine in the doublet (hsS1365, mmS1366) was compared.
  • Myocytes were transfected with plasmid encoding human WT TSC2, huS1364A TSC2, hsS1364E TSC2, hsS1365A TSC2, or hsS1365E TSC2.
  • TSC2 myocytes expressing either WT TSC2 or mutated TSC2 were exposed to ET1 (10 nM) for 48 hours, stimulating phosphorylation of TSC2 as detected by the antibody on when wild-type TSC2 protein was expressed.
  • Control cells were stimulated with vehicle for 48 hours.
  • the cell lysates were probed for phosphorylated TSC2 signal (Figure 3). Phosphorylation was observed when WT protein is expressed, but is equally blunted by site mutagenesis at either the first serine (hsS1364) or second serine (hsS1365).
  • Nppb pathological hypertrophy gene marker
  • SI 365 is equally effective in either attenuating (SE) or amplifying (SA) the hypertrophic signal stimulated in cardiac myocytes.
  • mTORCl signaling proteins p70 p70S6K and 4EBP1
  • markers of autophagy p62 and LC3-II
  • Myocytes expressing either WT TSC2 or mutated TSC2 were exposed to ET1 for 48 hours to induce hypertrophy, and cell lysates were examined for mTORCl signaling proteins and markers of autophagy ( Figure 5).
  • Endothelin 1 stimulates mTORCl activation depicted by increased phosphorylation of p70 p70S6K and 4EBP1), while also increasing both LC3-II and p62 - all consistent with mTORCl activation and stimulated autophagy.
  • hsS1364E or hsS1365E reduces mTORCl activation and p62, while further increasing LC3-II. This is consistent with enhanced autophagy and reduced growth stimulation.
  • expression of huS 1364A or huS 1365 A further increases mTORCl activation (more p70S6K and 4EBP1 phosphorylation, increased p62, reduced LC3-II).
  • Cells expressing hsS1365A-TSC2 displayed enhanced hypertrophy with ET1 exposure as compared to WT, and less green and red punctae consistent with reduced autophagy.
  • Cells expressing hsS1365E-TSC2 displayed much less hypertrophy with ET1 exposure as compared to WT or SA, and much more green and red puncae, indicating enhanced autophagy.

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Abstract

L'invention concerne des polypeptides du complexe 2 de la sclérose tubéreuse (TSC2) modifiés dans lesquels la capacité d'un résidu correspondant à un résidu sérine dans un polypeptide TSC2 de type sauvage à phosphoryler est modifiée. Dans certains aspects, un polypeptide TSC2 modifié ne peut pas être phosphorylé (<i />par exemple, en remplaçant le résidu sérine par un résidu alanine). Dans certains aspects, un polypeptide TSC2 modifié peut agir comme si elle était phosphorylée de manière constitutive (par exemple, en remplaçant le résidu sérine par un résidu d'acide glutamique). L'invention concerne également des cellules immunitaires modifiées comprenant des polypeptides TSC2 modifiés ou des séquences d'acide nucléique les codant, et des procédés de production et d'utilisation de telles cellules immunitaires modifiées.
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WO2019014624A1 (fr) 2017-07-14 2019-01-17 The Johns Hopkins University Tsc2 modifié
EP3652203A4 (fr) * 2017-07-14 2021-05-19 The Johns Hopkins University Tsc2 modifié
US11639508B2 (en) 2017-07-14 2023-05-02 The Johns Hopkins University Engineered TSC2
WO2023038816A1 (fr) * 2021-09-08 2023-03-16 Rigel Pharmaceuticals, Inc. Procédé de traitement d'une réponse immunologique excessive à une infection par un virus respiratoire

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